Kin Recognition in a Brooding Salamander Author(s): Brian S. Masters and Don C. Forester Source: Proceedings: Biological Sciences, Vol. 261, No. 1360 (Jul. 22, 1995), pp. 43-48 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/50045 Accessed: 03/03/2010 15:23 Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at http://www.jstor.org/page/info/about/policies/terms.jsp. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at http://www.jstor.org/action/showPublisher?publisherCode=rsl. Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission. JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact
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Kin recognition in a brooding salamander BRIAN S. MASTERS
AND
DON C. FORESTER
Departmentof Biological Sciences, Towson State University, Towson, Maryland 21204, U.S.A.
SUMMARY Egg guarding, or 'brooding', by the mountain dusky salamander (Desmognathusochrophaeus)is an example of maternal behaviour that can be experimentally manipulated and described quantitatively. It has been demonstrated that females of this species can specifically recognize, and will preferentially brood, their own eggs over those of a conspecific. We investigated whether this behaviour would extend to the selection of eggs of a more genetically similar animal in preference to those of a less similar animal, as might be predicted by inclusive fitness theory. We report here a highly significant correlation between time spent brooding and genetic similarity determined by random amplification of polymorphic DNA (RAPD) analysis. This is the first demonstration of a quantitative relation between genetic relatedness and maternal care in amphibians. Our findings have implications for the nature of maternal kin recognition in amphibians and its effect on kin selective behaviour in this class.
1. INTRODUCTION In all species of lungless salamanders (Plethodontidae) studied to date, the female parent remains with her eggs from oviposition until hatching (Bachmann 1964; Snyder 1971; Tilley 1972; Forester 1979a, Harris & Gill 1980; Juterbock 1987). Brooding females stop feeding and seldom venture from nest sites, except in response to large predators or seasonal flooding (Forester 1981, 1983; Juterbock 1987; Hom 1987). Brooding behaviour contributes to egg survival by reducing egg susceptibility to predation (Bachmann 1964; Snyder 1971; Forester 1978, 1979a), yolk layering (Forester 1979a), fungal infestation (Snyder 1971; Forester 1979 a), and desiccation (Forester 1984), but the cost to the female is high. Egg production and brooding have been estimated to represent 48 % of a female's annual energy budget (Fitzpatrick 1973). In addition, brooding exposes the female to a significant risk of predation, because most predators of eggs will also consume adult salamanders (Hom 1988). Female mountain dusky salamanders (Desmognathus ochrophaeus)frequently brood in close proximity to one another. Direct recognition of offspring has been observed in a number of different organisms living under conditions where parental care might be mistakenly directed toward the offspring of others (Beecher 1982; McCracken & Gustin 1991; Medvin et al. 1993), and has been demonstrated in D. ochrophaeus. When experimentally removed from nest sites, females of this species will return to brood their own eggs, even when their eggs are in close proximity to other unattended egg clutches (Forester 1979b). In laboratory discrimination experiments, females preferentially brood their own eggs over those of another female, in forced choice tests (Forester et al. 1983). Females can be induced, experimentally, to brood the eggs of another female (Tilley 1972; Forester Proc.R. Soc.Lond.B (1995)261, 43-48 Printedin GreatBritain
43
1979b). Forester (1979b) predicted that if clutch adoption occurs, females should favour kin over nonkin, as would be expected from inclusive fitness theory (Hamilton 1963; 1964a, b). To test this prediction, we used forced choice behavioural trials combined with random amplification of polymorphic DNA (RAPD) analysis to examine the effect of genetic similarity on brooding preference. RAPD is a relatively new polymerase chain reaction (PCR) based technique of DNA analysis that can be applied to a wide range of ecological problems (Hadrys et al. 1992; Black 1993). In this method, short oligonucleotides (typically eight-to-ten nucleotides long) are used to amplify anonymous portions of target genomes. The amplification products generated for different genomes are then compared, to estimate the genetic similarity of the genomes analysed. The technique has been used to examine genetic relatedness in a number of organisms, and similarity of RAPD generated fingerprints has been demonstrated, both theoretically and experimentally, to correlate with genetic similarity (Levitan & Grosberg 1993; Lewis & Snow 1993; Milligan & McMurray 1993; Lynch & Milligan 1994). 2. METHODS females were collected, along with Brooding D. ochrophaeus their eggs, from localities in western North Carolina. Preference trials were carried out in laboratory discrimination apparatuses as described by Forester et al. (1983). Females were placed in one of five rectangular discrimination apparatuses constructed from 0.5 cm aluminum sheet metal (inner LWH= 28 x 4.5 cm x 1.3 cm). Each apparatus presented females with a choice of two egg masses at the same developmental stage, positioned in simulated nesting chambers located at either end. Each chamber measured 4.5 cm x 4.5 cm (Lw), and the two chambers collectively comprised 32 % of the area available to the female within the ? 1995The Royal Society
44
B. S. Masters and D. C. Forester
Kin recognitionin a broodingsalamander
Table 1. Data generatedfor trials involvingeggs collectedless than 10 m or 100 m away from testfemale (BCI and BcII are designations of localities, each 10 m in diameter and 100 m apart. See ?2 for a discussion of how similarity indices (sis) were derived from comparisons of electrophoretic patterns of RAPD markers. The brooding index (brood index) was defined as the amount of time the female spent in one nesting chamber as a fraction of the total time she spent in either nesting chamber. Relative similarity values (dsi) are the arithmetic difference between the two si values obtained for each brooding trial (i.e. sI values derived from a comparison of the test female to the mother of each egg mass the test female could choose between). sI' and dsI' values are used in figure 2 only and reflect si values in which eight markers that were invariant in animals separated by a 100 m or less were not scored.) female BCI-l
BCI-3
BCII-11
BCII-2
egg choice
brood index
BC -2
0.83
BCI-13
BCI-14
dsi
SI'
dsi'
0.92 0.64
+0.28 -0.28
0.88
+0.45 -0.45
0.43
BCI-2
0.17
BC I-4
0.81 0.19
0.82 0.60
+0.22 -0.22
0.71
BCII-4 BCII-6
0.01
0.76
-0.14
-0.21
BC I-6
0.99
0.90
+0.14
0.62 0.83
BCII-3
0.60
0.89
0.40
0.73
+0.16 -0.16
0.80
BCI-2
+0.23 -0.23
0.33
0.57
+0.38 -0.38
+0.21
+0.06 -0.06
0.57
+0.11
0.46
-0.11
0.70
+0.10
0.50
0.60
-0.10
0.33
+0.17 -0.17
0.85
0.75
0.50
BCII-15
0.15
0.63
+0.12 -0.12
BCI-15
0.45
0.67
+0.11
0.55
0.55
+0.12 -0.12
0.40
BCII-16
0.29
-0.11
BCII-4
0.58
+0.19
0.67
BCI-3
0.42
0.82 0.63
-0.19
0.36
+0.31 -0.31
BCII-6
0.99
BC I-5
0.01
0.73 0.67
BCII-5
0.76
BC -4
0.24
BCI-14
BCII-5
BCII-4
sI
BCII-3
0.36
+0.14 -0.14
apparatus. Females were introduced into the centre of the apparatus through a cylinder and were allowed 30 min to acclimatize before being released. Each trial lasted 1440 min (24 h). To enter a nesting chamber, females had to pass through a pair of infrared light beams. Interruptions of light beams were recorded on the appropriate channel of an Esterline Angus event recorder enabling us to determine when females were present and absent from nesting chambers. Time spent in each nesting chamber was recorded for the 24 h trial period. Females were considered to exhibit significant brooding behaviour if they spent at least 763 min (53 % of the test period) in the nesting chambers, which was significantly (X2, p < 0.05) longer than the 461 min (32 % of
the test period) that would be predicted for random movement, because nesting chambers represented 32% of the total area available. Females were considered to show a significant brooding preference if they spent significantly more than 50 % of their brooding time (i.e. more than 70 %, x2, p < 0.05) with one clutch. In contrast to wild-type behaviour, females in 19 of the 58 behavioural trials spent little or no time brooding eggs. Data collected for females which failed to spend at least 53 % of the Proc. R. Soc. Lond. B (1995)
trial period within the nesting chambers were not included in our analysis because these females did not exhibit significant brooding behaviour. The percentage of the 24 h recording period that experimental females spent in either nesting chambers for trials listed in tables 1 and 2, ranged from 58 %-96 % (mean = 78 %). The brooding index was defined as: the amount of time the female spent in one nesting chamber as a fraction of the total time she spent in either nesting chamber. In the first experiment, females were given a choice of a clutch collected from within 10 m of their nesting sites, or a clutch collected 100 m away along the same stream. In the second experiment, females were given a choice between a clutch collected from within 10 m of their nesting sites, or a clutch collected from a separate river drainage 25 km away. No female was used in more than one trial. The genetic similarity of test females and mothers of test egg clutches was estimated by comparison of RAPD profiles. Comparisons of RAPDprofiles of test eggs were not used. It is not known if the egg-associated cues recognized by females are of maternal or embryonic origin. If the former is true, a comparison of females would be optimal, while if cues are of embryonic origin, a meaningful comparison would require analysis of all eggs in each clutch. We decided, in this study, to address the question of whether mothers were more likely to adopt the eggs of more genetically similar females over the eggs of less genetically similar females. This question was answered most directly by a comparison of females. DNA samples were isolated from the tips of tails of test females and mothers of test eggs using the procedure of Gustincich et al. (1991). RAPD analysis was carried out using
standard methodology (Williams et al. 1991) with modification (Masters 1995). RAPD reactions were repeated at least three times for all samples, and only consistently reproducible markers were scored. Four different tennucleotide primers (Operon Technologies, Alameda, California) were used to generate 32 different scored markers. Levitan & Grosberg (1993) made definitive maternity and paternity determinations for approximately two-thirds of progeny examined in marine hydrozoans using only four primers, but required 13 primers to assign parentage for all progeny. Lewis & Snow (1992) suggested scoring more than 50 markers for definitive paternity assignment. The number of markers employed in this study is insufficient to make definitive assignment of the level of genetic relatedness of all individuals examined (see Lynch & Milligan 1994), but it does allow for estimates of the relative genetic similarity of individuals. For example, comparisons of known siblings (embryos from the same clutch) to each other, and mothers to their progeny, revealed an average si (similarity index, see below) value of 0.711 for both groups in the BC population, whereas comparisonsof the pooled BC population revealed an average si value of 0.531 (unpublished data). Profiles of RAPDmarkersobtained by the electrophoresisof RAPD reaction products through 2 % agarose (by mass) were compared by using a similarity index (si) based on the formula: si = Nab(Na+ Nb)1, where Nab is the total number
of bands shared between the patterns of female a and female b, Na is the total number of bands in pattern a, and Nb is the total number of bands in pattern b (Nei & Li 1979). si values listed were obtained by a comparison of amplification products of DNA samples with regard to all 32 scored markers. Relative similarity values (dsI) are the arithmetic difference between the two SI values obtained for each brooding trial (i.e. sI values derived from a comparison of the test female to the mother of each egg mass the test female could choose between). Eight of the 32 scored markers were invariant in animals collected within a single stream (BC localities). si' and dsI' values were obtained by scoring only
Kin recognitionin a broodingsalamander B. S. Masters and D. C. Forester the remaining markersin comparisonsof animals from the BC localities alone. All DNA analysis was performedby one of us (B. S. M.) who had no knowledge of brooding trial outcomes. Technical problems prevented successful analysis of all females. DNA data were available for 20 trials in which females showed significant brooding behaviour, and for four trials in which such behaviour was not evident. These 24 trials represent a random subset of all behavioural trials.
V3o
.
o
*
0
c&Q
0.66
,0.* 0.2-
3. RESULTS Females showed significant brooding behaviour (by spending at least 53 % of the trial period in the nesting chambers, see ?2) in 18 of 33 trials involving withinstream experiments, and in 21 of 25 trials involving between-stream experiments. There was a significant correlation between geographical location and brooding preference in between-stream trials. When given the choice between eggs collected 10 m and 25 km from their nesting sites, 17 of 21 females showed a significant brooding preference (by spending more than 70 ?/ of total brooding time with a particular clutch, see ?2) for eggs from 10 m away (Wilcoxon Paired Sample Test, Z= -3.389, p < 0.001). There was, however, no such correlation in within-stream trials. When given a
oo *
0
45
/
*
- 0.6 - 0.4 - 0.2 0 0.2 0.4 0.6 relative similarity(dsi)
0.8
Figure 1. Linear regression of brooding index values as a function of relative similarity (dsi). Statistics: r = 0.88; r2= 0.75; p < 0.0001. Data from all experimental trials are plotted. 1.0 0
0.8 X -a 0.6 t
0
0
0 .0
Table 2. Data generatedfor trials involvingeggs collectedless than 10 m or 25 km away from test animals
0.4 0.2
(BCI, BCIII, CG, and si are designations of localities each
10 m in diameter. BCI and BcII are separated from CG and sI by 25 km.) female BCII1-2
BCIII-
BCII-5
CG-I si-2 BCIII-16 si-1 BCIII-17 BCIII-18 BCIII-19
BC
II-20
egg choice
brood index
si
dsi +0.31 -0.31
BCIII-3
0.96
0.67
si-3
0.04
0.36
BCIII-2 sI-l
0.99 0.01
0.60
BCIII-l
0.95
0.74
si-5
0.05
0.14
CG-2
0.99
0.70
BCIII-7
0.01
0.23
si-3
0.55
0.75
BCI-10
0.45
0.40
BCIII- 17
0.99
0.70
si-2
0.01
0.32
0.36
+0.24 -0.24 +0.60 -0.60 + 0.47 -0.47 +0.35 -0.35 +0.38 -0.38
si-2
0.98
0.75
+0.43
BCI-9
0.02
0.32
-0.43
BCIII-18 si-3
0.99
0.76
0.01
0.42
+0.34 -0.34
BCIII-19
0.92 0.08
0.80 0.18
+0.62 -0.62
BCII-20
1.0
0.91
+0.77
SI-5
-
0.14
-0.77
BCIII-16 sI-l
0.97 0.03
0.73
+0.44 -0.44
sI-4
Proc.R. Soc. Lond.B (1995)
0.29
-0.4
-0.2
0
0.2
0.4
0.6
relativesimilarity (dSI') Linear of 2. Figure regression brooding index values as a function of relative similarity in which eight markers, invariant in animals collected within the same stream, were not scored (dsi'). Statistics: r = 0.77; r2 = 0.59; p < 0.02. Data from experimental trials in which test females were given a choice between eggs collected 10 and 100 m away from their nesting sites (table 1) are plotted. choice of eggs collected 10 m and 100 m from their nesting sites, only 7 of 18 females showed a significant brooding preference for eggs from 10 m away (Wilcoxon Paired Sample test, Z=--1.285, p= 0.199). (Data not shown for brevity.) DNA data were obtained for 20 trials (11 betweenstream trials and nine within-stream trials) in which females spent 53 % or more of the trial period in nesting chambers (see tables I and 2). The correlation between brooding preference and the relative genetic similarity (dsI) of test animals was highly significant for this data set, which included both within- and between-stream trials (Spearman's r = 0.86, p < 0.001, figure 1). The correlation remains strong (Spearman's r = 0.77, p < 0.02) even when we only analyse the nine within-stream trials in which females were given a choice between eggs collected 10 m and 100 m from their nesting sites (see figures 2). A correlation between genetic similarity and brooding preference was not observed in experimental trials in which females showed no significant tendency to brood. In four trials in which females spent less than
46
B. S. Masters and D. C. Forester
Kin recognitionin a broodingsalamander
53 % of the 24 h trial period in the nesting chambers, and for which DNA data were obtained, no consistent relation between brooding preference and genetic similarity was evident (data not shown for brevity). In all four cases, test females showed no preference for the eggs of females with which they had a higher similarity index. 4. DISCUSSION The more parsimonious explanation of our data is that females are selecting eggs to brood by recognizing genetically determined cues associated with those eggs, rather than by using cues derived from environmental differences arising from microgeographical variation. Either mechanism could explain the observations obtained from our experiments involving betweenstream comparisons, but a mechanism relying on environmentally based cues becomes less plausible in light of the data obtained from within-stream comparisons. In these latter experiments, brooding did not correlate with geographical position, and it is only when genetic similarity data are considered that a statistically significant trend emerges. Such an observation is consistent with the highly specific nature of the maternal egg recognition ability previously demonstrated in this species. Females will preferentially brood their own eggs, even when given a choice between their eggs and eggs collected just a few centimetres from their nesting sites (Forester 1979b). One predicted cost of maternal recognition of kin is the risk of rejecting legitimate offspring (Beecher 1991). The egg selection mechanism of mountain dusky salamanders may minimize this cost. Willingness to brood in our experiments was apparently less strongly affected by the absolute value of the similarity index between the test female and the more similar clutch, than it was by the contrast in the two clutches of which she had a choice. This can be seen in a comparison of data from the two experiments. In the first experiment, in which females were given a choice between eggs collected 10 m and 100 m from their nesting sites (see table 1), the average maximal brooding preference was 77 o, the average maximal similarity index was 0.80 and the average difference between the two similarity indices was 0.15. In the second experiment, in which females were given a choice between eggs collected 10 m and 25 km from their nesting sites (see table 2), the average maximal brooding preference was 94?%, the average maximal similarity index was 0.74, and the average difference between the two similarity indices was 0.45. We propose that our observations can be explained by: (i) maternal recognition of genetically determined markers associated with candidate egg clutches; and (ii) adoption of the clutch with markers most closely corresponding to the female's own eggs. In the absence of an identical match, the female apparently prefers those eggs with the closest approximation of her recognition template, with willingness to brood increasing with the contrast of the choice offered. Slight errors in recognition, therefore, would not result in rejection of the female's own eggs and should still favour their selection over the eggs of other individuals. Proc.R. Soc. Lond.B (1995)
Preliminary evidence indicates that females nesting very close together are more closely related than would be predicted by chance (D. C. Forester & B.S. Masters, unpublished results). This arrangement would facilitate clutch adoption by close-kin should the appropriate combination of females and eggs be lost through predation or flooding. Perhaps more importantly, the presence of an elaborate recognition system might limit brood parasitism, a phenomenon that has never been reported in amphibians (Waldman 1991). Discrimination systems, based on olfaction (Forester 1986; Hepper & Waldman 1992) and using highly polymorphic markers, would be difficult to fool through mimicry. Marchetti (1992) has argued that, in birds, the cost of discrimination (i.e. mistaken rejection of one's own eggs) is so high that host defence mechanisms will be selected against, in the absence of significant levels of parasitism. This allows the evolutionary persistence of parasitism if the parasite is relatively rare or periodically switches between host species. Because the egg selection mechanism we propose for salamanders minimizes the cost of host defence by making the rejection of legitimate offspring unlikely, it may preclude brood parasitism as a viable reproductive tactic. Given the mechanism described here, the eggs of a distantly related animal (or those of another species) would be at a severe disadvantage, particularly in nests that cluster along kin-lines. It is interesting that the recognition system of D. ochrophaeusallows kin recognition, but does not provide species-specific recognition (Forester 1986). In laboratory brooding-preference experiments, D. ochrophaeus females that exhibited brooding behaviour showed no significant preference for conspecific eggs when given the choice between conspecific and congeneric (Desmognathusfuscus) eggs. However, this observation could result from the fact that recognition cues allowing individual discrimination must be highly polymorphic. As genetic distance increases, and markers on conspecific eggs become increasingly dissimilar, it is possible that a point is eventually reached where the markers presented by the eggs of a genetically distant conspecific are so different from the markers on a female's own eggs, that they are no more recognizable than those presented by congeneric eggs. Brooding preference of females when presented with such options would be expected to be random. This hypothesis could easily be tested by giving females a choice of eggs of varying genetic similarity. Females should prefer the eggs of close relatives over those of distant conspecifics or congeners, whereas they will show no preference between distant conspecifics and congeners. It has been proposed that many of the reports of apparent kin recognition might be a marginal, and even unintended, effect of species, group, or individual recognition (Grafen 1990). This has led to some debate as to what does, and what does not, constitute kin recognition, and it is important to consider the adaptive significance of any apparent kin recognition phenomenon (Blaustein et al. 1991; Grafen 1991). The existence of a sophisticated egg-recognition system in animals that rarely leave nesting sites is curious, but
Kin recognition in a broodingsalamander B. S. Masters and D. C. Forester 47 might be explained by the need for females to specifically locate their eggs after nesting site disruption. The patterns of adoption we see experimentally, in which females favour the eggs of more closely related animals over those of more distantly related animals, are possibly a by-product of an individual recognition system that allows for error. However, the fact that this by-product favours the progeny of more similar animals in the absence of an individual's own progeny, could have adaptive value. Adoption by brooding females of the eggs of other individuals has been demonstrated in field studies (Forester 1979b). It seems likely to us that adoptions under natural conditions may follow the kin-selective pattern observed in our laboratory trials. However this behaviour arose, it would have been adaptive for females to prefer the eggs of close relatives, if they invested in brooding eggs that were not their own. At this time, we can only speculate about the nature of the cues employed. Although the cues employed are probably detected by olfaction, we do not know whether they are maternally derived or arise from the embryos. It has been suggested that the highly polymorphic genes of the major histocompatibility complex (MHC) may be involved in individual-, and kin-recognition in a number of species (Brown & Eklund 1994). It seems very likely that whatever cues are employed by the recognition system described here, the specificity demanded, requires the variability provided by the MHC or some other comparatively polymorphic genetic loci. We thank L. S. Johnson for many valuable comments on the manuscript. This work was supported, in part, by the Towson State University Office of Research Administration and The Highlands Biological Station in Highlands, North Carolina. REFERENCES Bachmann, M. D. 1964 Maternal behavior of the redbacked salamander, Plethodoncinereus.Ph.D. dissertation, University of Michigan. Beecher, M. D. 1982 Signature systems and kin recognition. Am. Zool. 22, 477-490. Beecher, M. D. 1991 Successes and failures of parent(ed. P. offspring recognition in animals. In Kin recognition G. Hepper) pp. 94-124. Cambridge University Press. Black, W.C., IV 1993 PCR with arbitrary primers: approach with care. Insectmolec.Biol. 2, 1-6. Blaustein, A. R., Berkoff, M., Byers, J. A. & Daniels, T.J. 1991 Kin recognition in vertebrates: what do we really know about adaptive value? Anim.Behav.41, 1079-1083. Brown, J. L. & Eklund, A. 1994 Kin recognition and the major histocompatibility complex: an integrative review. Am. Nat. 143, 435-461. Fitzpatrick, L. C. 1973 Energy allocation in the Allegheny Mountain salamander, Desmognathusochrophaeus.Ecol. Monogr.43, 43-58. Forester, D. C. 1978 Laboratory encounters between (Amphibia, Urodela, attending Desmognathusochrophaeus Plethodontidae) females and potential predators. J. Herpetol.111, 311-316. Forester, D. C. 1979a The adaptiveness of parental care in (Urodela: Plethodontidae). Copeia. ochrophaeus Desmognathus 1979, 332-341. Proc.R. Soc.Lond.B (1995)
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