Behav Ecol Sociobiol (2000) 47:180–187
© Springer-Verlag 2000
O R I G I N A L A RT I C L E
Michelle L. Hall · Robert D. Magrath
Duetting and mate-guarding in Australian magpie-larks (Grallina cyanoleuca)
Received: 10 May 1999 / Received in revised form: 27 September 1999 / Accepted: 2 October 1999
Abstract A recently favored hypothesis is that duetting in birds has a mate-guarding function: a male responds vocally to his partner’s song, thereby forming a duet that repels males who are attracted to her song. Previous studies have not provided unambiguous tests of the mate-guarding hypothesis because: (1) the probability of a male answering his partner’s song has not been shown to increase specifically when the female is fertile, and (2) the probability of a male answering his partner’s song has not been assessed separately from simply a higher song initiation rate. We investigated extra-pair paternity, mate-guarding, and duetting in the socially monogamous Australian magpie-lark (Grallina cyanoleuca). DNA fingerprinting revealed that 3% of young were the result of extra-pair paternity, and we found that males guarded fertile females by staying close to them. However, males did not initiate songs at a higher rate when females were fertile and actually reduced their probability of replying to female song during this period. We conclude that although male magpie-larks did guard fertile females in an attempt to prevent extra-pair copulations, they did not use duetting for this purpose. Key words Bird song · Duet · Mate-guard · Monogamy · DNA fingerprinting
Introduction In many bird species, song is restricted to males, and functions primarily in mate attraction and territorial defense (Searcy and Andersson 1986; Catchpole and Slater Communicated by W.A. Searcy M.L. Hall (✉) · R.D. Magrath Division of Botany and Zoology, Australian National University Canberra, A.C.T., 0200, Australia e-mail:
[email protected] Tel.: +61-2-62492866, Fax: +61-2-62495573
1995), but there are species where females sing too, and their song often has similar functions (Langmore 1998). Males and females of some species combine their songs to produce precisely coordinated acoustic displays called duets. Many functions have been suggested for duetting, most reflecting the idea that duets play a role in the establishment and maintenance of pair bonds, territories, or both (reviews in von Helversen 1980; Farabaugh 1982; Ritchison 1983). Hypotheses that duetting allows partners to maintain contact when out of sight (Thorpe 1963), or stimulate reproductive behavior in the female (Sonnenschein and Reyer 1983) imply that duetting is a cooperative display for within-pair communication. Alternatively, duetting might be directed by the pair at other birds to advertise and defend their territory (Wiley and Wiley 1977). More recent hypotheses have suggested that duetting is a result of independent male and female strategies: a “coy” partner demanding investment in learning a pair-specific duet as a prerequisite for mating (Wickler 1980), or an individual assessing its partner’s commitment by its probability of answering its partner’s song (Smith 1994). Testing hypotheses for the function of duetting is complex in part because duets could have more than one function in a species, and also because the functions of duetting could differ between species. We focus on one hypothesis which has received recent support – that duetting is used for mate-guarding and results from conflict rather than cooperation between the sexes (Sonnenschein and Reyer 1983; Smith 1994; Levin 1996). In this case, duets occur when males give a temporally coordinated response to their partners’ song, and function to repel other males attracted to female song (Stokes and Williams 1968; Levin 1996). We use the term “mate-guarding” in the sense of guarding paternity, rather than in the wider sense of guarding the “pair bond”. Males are more vulnerable to loss of parentage than females and may use a variety of mate-guarding strategies, including staying close to the female, copulating frequently, and increasing territorial behavior and song rates (Birkhead and Moller 1992). The effectiveness of paternity-guards depends on female behavior. Close following or
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frequent copulations are likely to reduce extra-pair paternity even if females actively pursue extra-pair copulations, whereas territorial behavior and high song rates are probably less effective because they are directed at intruding males (Lifjeld et al. 1994). Nevertheless, male song rate in many species peaks during the female’s fertile period (Moller 1991). This may serve to reduce intrusions by rival males (Sheldon 1994), and may also stimulate reproductive behavior in females (Kroodsma 1976). In duetting species, males could guard females acoustically in two ways: they could be more responsive to female song, forming duets, or they could simply initiate songs at a higher rate (Sonnenschein and Reyer 1983). Duets in bay wrens (Thryothorus nigricapillus) resulted from male responsiveness to female song (96% of duets in this species were initiated by females), and males were attracted to playback of female song, consistent with a mate-guarding function (Levin 1996). However, evidence for the mate-guarding hypothesis for duetting is incomplete because a key prediction, that a male’s probability of answering his partner’s song increases specifically when she is fertile, was not explicitly tested. We tested whether mate-guarding was one of the functions of duetting in the Australian magpie-lark (Grallina cyanoleuca). Magpie-lark duets are believed to function primarily to maintain the pair bond, and secondarily in territory maintenance (Tingay 1974), but the various ways in which duets might achieve these ends have yet to be established. Both males and females can initiate duets (females initiate about 40% of duets), and both can also sing solo songs (Tingay 1974), allowing us to distinguish the two potential forms of acoustic paternity-guards – song initiation rate and responsiveness to a partner’s songs. Magpie-larks are socially monogamous and pairs defend all-purpose territories throughout the year. Males invest heavily in parental care, sharing nest-building, incubation and feeding duties approximately equally with females (Hall 1999), and they follow fertile females closely (Neill and Lill 1998). We used DNA fingerprinting to determine the frequency of extra-pair paternity, and investigated whether males in this population guarded paternity by maintaining proximity or copulating frequently. We also measured song initiation rates and probability of answering partner’s songs to determine whether either song initiation rate or duetting was used to guard paternity.
Methods Study species and population Magpie-larks are passerines that are closely related to the monarchs, in the subfamily Dicrurinae (Sibley and Ahlquist 1990). They are found throughout Australia in open habitats (Schodde and Tidemann 1986). Males and females are easily distinguishable on the basis of plumage differences (Disney 1974). Duets are audible from neighboring territories, and are sung throughout the year, peaking seasonally at the start of the breeding season, and daily at dawn (Tingay 1974). Although male and female contributions to duets are not obviously sex-specific, individuals can distinguish between recorded song of males and females (M.L. Hall,
unpublished work). We observed a population of magpie-larks resident on the campus of the Australian National University from August to December in 1995 and 1996. The study area was approximately 0.5 km2, and contained 19–24 territories. Pairs were marked with colored leg bands to allow individual recognition. Analysis of parentage Blood samples for DNA fingerprinting were taken from 21 pairs and their offspring over the 2 breeding seasons. Offspring were bled as nestlings at 11–14 days old if the nest was accessible or, if not, while still on the natal territory after leaving the nest. Blood samples of 50–200 µl were collected by puncturing the brachial vein and were then stored at –20°C. We extracted DNA from blood samples by the NaCl-salt method for avian blood (Bruford et al. 1992), and stored it in 1x TE buffer at –20°C. DNA samples were digested with restriction enzyme HaeIII, and 6 µg of digested DNA were electrophoresed on 40 cm 0.8% agarose fingerprint gels at 70 V for about 70 h. Following electrophoresis, DNA was transferred to Hybond-N membranes by Southern blotting, and then baked at 80°C for 2 h. Membranes were hybridized with radioactively labelled minisatellite probe per (Shin et al. 1985), and some were subsequently hybridized to radioactively labelled 33.6 (Jeffreys et al. 1985) to produce autoradiographs. _ We scored 11.3±3.37 (X±SD) bands per individual (n=145) in the 21 families probed with per, and 10.0±2.61 bands per individual (n=65) in the 10 families probed with Jeffreys 33.6. DNA fragments detected by different probes may not be independent (Westneat 1990), so results from the two probes were assessed separately. We compared DNA fragments (bands) of offspring with putative parents to assess parentage. To facilitate comparisons, family groups were run on the same gel in adjacent lanes, with nestlings on either side of putative parents. We scored bands not more than 0.5 mm apart with not more than a twofold intensity difference as the same. To assess parentage, we identified novel bands (bands present in offspring, but not in either of the putative parents), and calculated the proportion of bands shared between offspring and each of their putative parents (Wetton et al. 1987). We calculated the proportion of bands shared between members of breeding pairs as a measure of background band-sharing. Behavioral observations We watched 13 pairs for 30-min periods in the mornings, 2–5 h after sunrise, over the course of the breeding season in 1995, collecting information on copulations, proximity, and movements, relative to the start of egg-laying. We estimated the distance between pair members at 1-min intervals, and calculated the proportion of time that pair members were within 5 m of one another. To determine who maintained proximity we counted movements of more than 5 m by each individual relative to its partner – either “away” from the partner or closer to it. A move closer to the partner was classified as a move “towards”, unless it occurred within 20 s of a move away by the partner, in which case it was used to calculate the proportion of partner’s moves away that were “followed”. If an individual’s partner did not move away from it during the observation period, no measure of following was obtained. As an overall measure of maintenance of proximity, we calculated the proportion of all movements by an individual which brought it closer to its partner (i.e., moves towards and follows). We did not continue observations after incubation started, on the day the second egg was laid, as either the male or the female was on the nest at all times. We subsampled the data for analysis, comparing “non-fertile” and “fertile” periods. “Non-fertile” period watches were taken from before nest-building started in most cases (or over 16 days before the first egg was laid), when females were unlikely to be fertile. Since the “fertile” period of magpie-larks was not known, we were conservative and used watches as close as possible to the day the first egg in a clutch was laid, when females were most likely to be fertile (watches used ranged from 1–4 days before the first egg).
182 In the 1996 breeding season we recorded the vocalizations given by each member of 13 pairs in recording sessions during non-fertile and fertile periods, as defined above. Recording sessions started before sunrise with the first, or close to first, vocalizations of the morning, and lasted on average 35 min. Copulations occurring during these observation periods were noted. Vocalizations were recorded using a Sony digital audio tape-corder (DAT) TCD-D10 Pro II and a Sennheiser MKH 416T directional microphone and, where possible, the identity of the singer and the distance between pair members were noted. We identified the singer in the field for an average of 67% of vocalizations (range 18–98 vocalizations per recording session), and used sonagraphic analysis to identify individually distinctive song types, so that on average the singer was identified for 76% of vocalizations in a recording session (minimum 40%). We compared song initiation rates and probability of answering partner’s song for males and females during the non-fertile and fertile periods using only those vocalizations for which the singers were identified. The “song initiation rate” calculated for each individual consisted of all the solo songs and duets initiated by it in 30 min. “Probability of answering a partner’s song” was the proportion of its partner’s song initiations to which an individual responded with temporally coordinated song to form a duet. For example, the probability of a female answering her partner’s song was calculated as: (number of duets initiated by male)/(number of male solos+number of duets initiated by male). We used the subsample of songs for which distance between pair members was recorded to investigate the effect of distance on the probability of males and females answering their partner’s song in non-fertile and fertile periods. Statistical analyses To determine whether males guarded fertile females, we compared male behavior in fertile and non-fertile periods. We measured the effect of breeding stage on female behavior as a further control against which to assess changes in male behavior. Repeated measures analysis of variance was used to analyze data when the assumptions relating to normality and variance were met by either the original or transformed data. In these analyses, pairs were treated as subjects, and sex and stage of the breeding cycle as within-subject factors. For data that did not satisfy the assumptions of repeated measures analysis of variance, we used Wilcoxon’s matched-pairs signed-ranks tests. The statistical package SPSS 6.1 (Norusis 1994) was used for computation. To assess the effect of intrapair distance on the probability of answering a partner’s song, we used generalized linear mixed modeling, a procedure that allows analysis of unbalanced data with a binomial error distribution (response to song initiation or not). In addition to fixed effects, these models incorporate random effects which account for the dependence associated with multiple sampling from pairs. The GLMM procedure (Welham 1995) in the statistical package Genstat 5 release 3.2 (Genstat 1993) was used to fit models according to the method of Schall (1991) using a logit link function. Pair was treated as a random effect, and sex, stage of the breeding cycle, and intrapair distance as categorical fixed effects. The significance of fixed effects and interactions between them were assessed using Wald’s statistics calculated when the term of interest was fitted last in the model.
Results Analysis of parentage DNA fingerprinting with the minisatellite probe per revealed that 3 of 103 (3%) offspring, in 3 of 47 broods (6%), were not related to their putative father. Up to 4 novel DNA fragments per individual were detected by the per probe (n=1 individual with 2, 3, and 4 novel
Fig. 1 Relationship between the number of novel bands present in offspring and band-sharing coefficients [(2Nab/(Na+Nb)] (Wetton et al. 1987) with a their putative father, and b their putative mother
bands respectively, n=5 with 1 novel band, and n=95 with 0 novel bands). Unlike the other offspring, the three offspring with more than one novel band all had low band-sharing coefficients with their putative father (Fig. 1a), although all offspring had high band-sharing coefficients with their mother (Fig. 1b). This suggested that up to one novel band per individual was probably due to mutation, but additional novel bands indicated that offspring were the result of extra-pair matings by the female rather than egg-dumping. Probing a subsample of families with Jeffreys 33.6 confirmed the results from per. Four of 45 offspring had novel bands – a single novel band was detected in 3 individuals (per had detected a single novel band in 1 of these individuals), and 3 novel bands were detected in the other individual (4 novel bands detected by per). Again, the latter individual was distinguished from other offspring because it shared no bands with its putative father, and 0.5 of its bands with its putative mother. The other two cases of extra-pair paternity detected by per were not among the subsample scored with Jeffreys 33.6. Offspring with one or no novel bands were assumed to be descendants of both putative parents. In these offspring, band-sharing coefficients with their mothers were no different to band-sharing coefficients with their fathers – when probed with per (X±SD=0.56±0.10 bands shared with the mother, 0.54±0.10 with the father; paired t-test
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Fig. 2 Frequency distribution of band-sharing coefficients of offspring with their putative fathers (black bars), offspring with their putative mothers (unfilled bars), and breeding pairs, assumed to be unrelated (hatched bars)
– t99=0.99, P=0.33), or Jeffreys 33.6 (X±SD=0.59±0.12 bands shared with the mother, 0.57±0.12 with the father; paired t-test t43=0.63, P=0.53). Except for the three cases of extra-pair paternity, the frequency distributions of band-sharing coefficients of offspring with their putative father and mother were similar, and higher than bandsharing coefficients between parents, who were presumed to be unrelated to one another (Fig. 2). Band-sharing coefficients between pair members therefore gave an indication of background levels of band-sharing – (X±SD=0.18±0.08, n=21 pairs for per and 0.19±0.17, n=10 pairs for Jeffreys 33.6). Proximity and copulation rate Pair members spent more time close together when the fe– male was fertile than when she was not fertile (X±SE proportion of time within 5 m=0.56±0.06 in fertile period, 0.30±0.05 in non-fertile period; two-tailed paired t-test: t12=–4.87, P
