Journal of Ornithology

, Volume 149, Issue 4, pp 521–527

Offspring sex ratio in the sequentially polygamous Penduline Tit Remiz pendulinus


    • Department of Biology and BiochemistryUniversity of Bath
    • Animal Ecology Group, Centre for Ecological and Evolutionary StudiesUniversity of Groningen
  • Jan Komdeur
    • Animal Ecology Group, Centre for Ecological and Evolutionary StudiesUniversity of Groningen
  • Marco van der Velde
    • Animal Ecology Group, Centre for Ecological and Evolutionary StudiesUniversity of Groningen
  • István Szentirmai
    • Department of EthologyEötvös Loránd University
    • Őrsèg National Park
  • Xutong Yang
    • Department of Biology and BiochemistryUniversity of Bath
  • Richard Ffrench-Constant
    • Department of Biology and BiochemistryUniversity of Bath
    • Centre for Ecology and ConservationUniversity of Exeter
  • Tamás Székely
    • Department of Biology and BiochemistryUniversity of Bath
Original Article

DOI: 10.1007/s10336-008-0299-5

Cite this article as:
van Dijk, R.E., Komdeur, J., van der Velde, M. et al. J Ornithol (2008) 149: 521. doi:10.1007/s10336-008-0299-5


Despite the growing literature on facultative sex-ratio adjustment in chromosomal sex-determining vertebrate taxa (birds, mammals), the consistency of results is often low between studies and species. Here, we investigate the primary and secondary offspring sex ratio of a small passerine bird, the Eurasian Penduline Tit (Remiz pendulinus) in three consecutive years. This species has a uniquely diverse breeding system, in which the male (and/or the female) abandons the nest during egg-laying, and starts a new breeding attempt. This allowed us to test (1) whether patterns of parental care, i.e., male-only care, female-only care or biparental desertion, influence offspring sex ratio, and (2) whether the offspring sex ratio is repeatable between successive clutches of males and females. Using molecular markers to sex 497 offspring in 176 broods, we show that (1) offspring sex ratio does not depend on which parent provides care, and (2) the offspring sex ratio is not repeatable between clutches of a given individual. The overall primary and secondary offspring sex ratio at a population level is not different from parity (54 ± 6% males, and 50 ± 3% (mean ± SE), respectively). We suggest that ecological and phenotypic factors, rather than individual traits of parents, may influence offspring’s sex, and conclude that there is currently no evidence for a facultative adjustment of offspring sex ratio in the Penduline Tit.


Parental careBreeding systemRemiz pendulinusRepeatabilitySex allocation


Fisher’s (1930) frequency-dependent model of sex allocation predicts that natural selection will maintain an even population sex ratio as long as the cost of producing a male is equal to that of producing a female. However, the optimal offspring sex ratio of an individual parent may deviate from parity due to a variety of factors resulting in a relative fitness difference between sons and daughters (Trivers and Willard 1973; Hasselquist and Kempenaers 2002; Komdeur and Pen 2002; West et al. 2002). The recent advent of molecular sexing techniques has contributed significantly to the question whether mothers should bias the sex ratio of their offspring. In contrast to previous work (Charnov 1982; Clutton-Brock 1986), recent studies suggest that animals with chromosomal sex determination, such as birds and mammals, are indeed able to facultatively adjust the primary sex ratio of their offspring in an adaptive manner (Komdeur and Pen 2002; Hasselquist and Kempenaers 2002; West et al. 2002; West and Sheldon 2002). For example, females may bias the sex ratio of their offspring in response to the attractiveness of the father (Kempenaers et al. 1997; Sheldon et al. 1999), the timing of breeding (Daan et al. 1996; Badyaev et al. 2003; Székely et al. 2004), levels of parental care (Clutton-Brock 1991), and to biased population sex ratios leading to more intense competition for mates among one sex (Hamilton 1967). However, despite several studies reporting biases in offspring sex ratio, others failed to find deviations from parity (e.g., Saino et al. 1999; Grindstaff et al. 2001; reviewed in Komdeur and Pen 2002). This underlines the need for further studies including those reporting non-significant effects as well as significant findings to avoid publication bias (Festa-Bianchet 1996; Cockburn et al. 2002; Hasselquist and Kempenaers 2002).

Griffin et al. (2005) argue that across species the selection on sex-ratio adjustment may be variable due to differences in breeding system, sexual dimorphism, and life-histories, and as such cause biological variation in sex-ratio adjustment, whereas within species such variability in selection is less straightforward. Studies investigating the repeatability of facultative sex-ratio adjustment in the same species between years, and for the same individuals are scarce, yet crucial to determine with confidence the frequency of sex-ratio modification in specific taxa (Palmer 2000; Ewen et al. 2004; Cassey et al. 2006; Korsten et al. 2006).

Here, we investigate sex allocation in the Eurasian Penduline Tit (Remiz pendulinus) in three consecutive breeding seasons. The Penduline Tit is a small passerine (body mass about 9 g) with modest sexual dimorphism: adult males have brighter plumage and larger eye-stripes than females (Cramp et al. 1993; Glutz von Blotzheim and Bauer 1993; Kingma et al. 2008). Penduline tits have highly diverse breeding strategies that involve sequential polygamy by both sexes, and uniparental incubation and subsequent brood care by either the male (5–20% of nests) or the female (50–70% of nests). An unusual feature of Penduline Tit breeding biology is the high frequency of nest desertion by both parents (approximately 30% of clutches) dooming these eggs to failure (Franz 1988; Persson and Öhrström 1989; Szentirmai et al. 2007). Since desertion takes place during egg laying, clutches cared for by the male and clutches deserted by both parents are usually smaller (3.4 ± 1.3 eggs; mean ± SD) than clutches cared for by the female (5.9 ± 1.3 eggs), since the female may lay a few more eggs after her mate has deserted (Franz 1991; Persson and Öhrström 1989).

The breeding system of Penduline Tits allowed us to address three major objectives. Firstly, we tested whether offspring sex ratio depends on which parent provides care, by comparing offspring sex ratios of male-only cared clutches with those in female-only cared and biparentally deserted clutches. The sex of the offspring may vary with laying order in several species (e.g., Kilner 1998; Komdeur et al. 2002; Cichoń et al. 2003), and given that desertion takes place during egg-laying in Penduline Tits, this may lead to a different sex ratio in male-only cared and female-only cared clutches. Since the proportion of sons has been reported to increase with laying order (Kilner 1998; Krebs et al. 2002), we expected female-only cared clutches to be more male-biased than male-only cared clutches in Penduline Tits, since females usually lay two to three more eggs after the male deserts.

Secondly, we expected repeatable sex allocation between successive nests of a given individual if individual characteristics, such as attractiveness or parental abilities, influence the offsprings' sex, and is independent of the quality of the mate, territory and season (but see Oddie and Reim 2002). To investigate how variation in brood sex ratio may depend on individual characteristics, we calculated the repeatability of offspring sex ratio of individual males and females that produced several broods in a given season.

Thirdly, we tested whether offspring sex ratio deviated from unity at the population level and whether the distribution of sons was different from binomial distribution. The rationale behind the latter was that even if offspring sex-ratio is not different from unity at population level, some females may produce largely sons and others largely daughters, and deviation of these extreme phenotypes may be different from the binomial expectation (e.g., Radford and Blakey 2000; Westneat et al. 2002; Dietrich-Bischoff et al. 2006).



We studied the Penduline Tits at an extensive system of fish-ponds near Szeged in southern Hungary (46°19′N, 20°5′E) where they breed along the dikes which separate the ponds. Fieldwork was carried out between April and August 2002–2004. Totals of 214, 183, and 178 nests were found in 2002, 2003, and 2004, respectively. Males start building their nest and sing to attract a female, although it takes 8.9 ± 7.0 days for a male to find a mate (n = 111 males). Of all nests, 52% was abandoned either before pair formation had taken place [i.e., the male was unsuccessful in attracting a female (37%), or the nest was abandoned due to disturbance by humans, heavy winds or predation (12%), or a new owner overtook the nest (4%)]. We searched the study site to identify unpaired, nest-building males, and then visited the males every other day to monitor their status by observing them for at least 15 min (see details in Bleeker et al. 2005; Van Dijk et al. 2007).

We collected the following data at each nest:
  1. 1.

    Nest initiation date. The exact date of initiation could be determined for nests found when only a small amount of nest material is woven around a twig (categorized as stage A; see Fig. B on p. 385 in Cramp et al. 1993). Initiation dates for nests found in later stages (stages B–E; see Fig. C–I on pp. 386–387 in Cramp et al. 1993) were estimated by comparison with the progress of nests that had been followed continuously since stage A (Szentirmai et al. 2005).

  2. 2.

    Date of pair formation. A male was considered paired if he was seen copulating with a female near the nest, or the pair was observed building the nest together.

  3. 3.

    Sex of attendant parent. We identified which parent was attending the nest at each stage of the nesting cycle (nest building, egg-laying, incubation, nestling period). A parent was considered to have deserted the nest if it had not been seen during at least two consecutive nest checks. Birds classified as ‘deserted’ were never seen at the nest subsequently.

  4. 4.

    Start of incubation. This was determined by observing the behavior of the parent: incubating parents stay inside the nest for longer continuous periods then nest building birds.


Adults were individually marked using a unique combination of three color rings, and one numbered metal ring from the Hungarian Ornithological Institute. Returning rates of adults are low across years: out of 195 color-ringed males from 2002 to 2003, only 13 males were resighted in one or both subsequent years. Similarly for females: out of 87 color-ringed females, only 8 were resighted. A small blood sample (about 10 μl) was taken from adults and 10-day-old nestlings by puncturing their brachial vein. Unhatched eggs in all clutches including the incubated ones were checked for the presence of an embryo. Clutches deserted by both parents were taken to the laboratory and incubated indoors using an incubator set at 37.5°C. Eggs were opened after being incubated for 5 days and any visible embryos were placed in an Eppendorf tube.

Molecular sexing

For nestlings, DNA extraction was carried out using the GenomicPrep Blood DNA Isolation Kit (Amersham Biosciences, USA). DNA from egg-samples was extracted using the Chelex method (Walsh et al. 1991). The sex of the offspring was determined by DNA amplification using P2 and P8 primers for PCR under the reaction conditions given in Griffiths et al. (1998). We blindly repeated the molecular sexing of 26 nestling and 14 egg samples chosen at random: all matched the sex assigned by the first test. Using DNA collected in 2005, we also compared the molecular sexing of 22 adults (14 males, 8 females) with the sexing done in the field based on plumage and behavioral characteristics. All adults were sexed consistently with our field observations.

Statistical analyses

We investigated repeatability of offspring sex ratios for males and females using bootstrapping, since parametric estimation (Lessells and Boag 1987; Harper 1994) was not feasible given that the proportion of sons was not normally distributed and the variances were heteroscedastic. We calculated the within-year repeatability of offspring sex-ratio by choosing males (or females) that had multiple nests in a given season. We only include within-year repeatabilities and not between years, because returning rates are low (see above). First, we calculated the absolute mean difference in the proportion of sons between all nests of a given parent, and took the mean of these individual means (δ test statistic). Second, the sex of the offspring was randomized 104 times by keeping the original data structure. At each iteration we calculated δ as for real data. Third, we calculated the proportion of cases in which the randomized values were less than the test δ. We report the test statistic and the probability of finding a value smaller than or equal to the test δ. Randomization was carried out by Resampling Stats™ for Excel version 3.2 (2006).

All analyses were carried out using three sets of data. First, we used the full dataset that included all nestlings and unhatched eggs. Second, we repeated the analyses separately after dividing the dataset into eggs and nestlings. We provide the results of both given the interest in primary (eggs) and secondary (nestlings) sex ratios. Third, we investigated the influence of which parent, male, female or none, provided parental care on offspring sex ratio. If several nests were available for an individually marked male or female, we selected one nest randomly to avoid pseudoreplication, under the condition that male-only cared nests were selected in priority to female-only or biparentally deserted nests due to the limited number of male-only cared nests (12.4% of nests). At 42 of randomly selected nests of individually marked males the female was unringed. At 18 of these nests multiple females bred at the same time so these clutches were produced by different females. For the remaining 24 nests where the female was unringed pseudoreplication cannot be excluded, although we suspect it is small given (1) the size of our breeding population (see above), (2) the fact that offspring sex ratio is not repeatable between nests of given individuals (see "Results"), and (3) mate fidelity is low and remating between adult breeders is extremely rare.

We used generalized linear mixed models (GLMMs) with binomial error distribution and a logit link function to test the influence of parental sex on offspring sex ratio of individual eggs and nestlings nested within a brood using R (R Development Core Team 2005). The GLMM used the sex of each egg or nestling as the unit of analyses, the type of parental care (male-only, female-only or biparental desertion) as the explanatory variable, and brood identification (ID) as a random factor. The dispersion parameter was set to 1.0. Date of pair formation and year were also included in the model to test for an influence of season or year on the effect of parental care type. To assess the influence of parental care we used the Wald statistic, which has an approximately χ2 distribution. We considered using parent ID as a random factor in the analyses, but rejected this idea because for many broods the parents were not ringed (in 47% of 176 broods, the male ID, female ID or both were unknown). More females than males were unringed, and unringed females were especially common in male-only cared and biparentally deserted nests, since females were usually trapped during incubation.

At the population level, we tested whether the proportion of sons deviates from 0.5 using a one-sample Wilcoxon signed-ranks test in MINITAB® release 12.2 (2004). To test whether the offspring sex ratio deviates from the binomial distribution (Sokal and Rohlf 1995), we calculated the number of males for all nests with equal number of sexed eggs and/or chicks and compared the observed frequencies with the expected ones using a χ2 test. Nests with between two and six sexed eggs and/or chicks were included in the latter analyses.

Data are represented as means ± SE, and we provide two-tailed probabilities. Statistical significance was judged at the 0.05 level.


The attendant father was identified at 95 out of a total of 176 nests sampled for offspring sex determination. The attendant mother was known at 85 out of the 176 nests. Of those known males, 41 produced several broods within a year (mean 2.41, range 2–5 broods), compared with 19 individually marked females (mean 2.21, range 2–4 broods). We sampled 64 nests in 2002 (a total of 24 eggs and 152 nestlings), 55 nests in 2003 (37 eggs and 104 nestlings), and 57 nests in 2004 (36 eggs and 144 nestlings); in total, 497 offspring (97 eggs and 400 nestlings) in 176 nests. These included both partial and complete clutches and broods (Fiala 1980). For 57 nests, only eggs were analyzed, of which 8 were complete and 49 partial clutches. For 115 nests, only nestlings were analyzed, of which 88 were complete and 27 partial broods. For 4 nests, both eggs and nestlings were analyzed. Of each of these nests, a sub-sample of the full clutch or brood was included in the analyses.

Offspring sex ratio and parental care

Out of 169 nests, 21 (12.4%) were cared for by the male, 103 (60.9%) by the female, and 45 (26.6%) nests were deserted by both parents. At seven nests, the sex of the attendant parent was not known. Offspring sex ratio did not differ between male-only care, female-only care and biparentally deserted nests, when both eggs and nestlings were included in the analyses (51 ± 7, 42 ± 4, 50 ± 10%, respectively; χ2 = 2.04, df = 2, P = 0.36, n = 247 offspring in 90 broods). This result remained consistent when the analysis was restricted to nestlings, thereby, per definition, excluding biparentally deserted nests: 56 ± 7%, 43 ± 4%, respectively; χ2 = 1.11, df = 1, P = 0.29, n = 202 offspring in 62 broods. We did not find an effect of year or date of pair formation when these were incorporated into the model (P > 0.31).


The proportion of sons was not different from random between nests of a given male (51 ± 4%; δ = 0.35, P = 0.14, n = 37 males), or of a given female (40 ± 6%; δ = 0.45, P = 0.76, n = 15 females; nests include both eggs and nestlings). Given that there was no effect of parental care on offspring sex ratio (see above), we did not control for a potential effect of parental care on repeatability. Sample sizes did not allow us to analyse eggs and nestlings separately.

Offspring sex ratio

A total of 252 out of 497 offspring (51%, pooling unhatched eggs and nestlings) were male. Of the 97 unhatched eggs, 52% were male, compared to 51% of 400 nestlings; therefore, the overall sex ratios did not differ from parity (Table 1a). The latter results stand when the analysis was restricted to the average proportion of sons of individually marked males (Table 1b) or females (Table 1c), suggesting that pseudoreplication unlikely influenced the results.
Table 1

Percentage of sons in Eurasian Penduline Tits (Remiz pendulinus) in all samples (a), and the percentage of sons of individually marked males (b), and of individually marked females (c)


% of sons (mean ± SE)



n (nests)


Eggs only

54 ± 6




Nestlings only

50 ± 3




Eggs and nestlings

52 ± 3





% of sons (mean ± SE)



n (males)


Eggs only

45 ± 8




Nestlings only

47 ± 3




Eggs and nestlings

46 ± 4





% of sons (mean ± SE)



n (females)


Eggs only

38 ± 12




Nestlings only

53 ± 3




Eggs and nestlings

51 ± 3




aTest statistic of Wilcoxon one-sample tests using 50% as test-median. Sex ratio per nest is used as datum

Offspring sex ratios were not different from binomial distribution (brood size = 2, n = 38 nests, P = 0.623; brood size = 3, n = 33, P = 0.424; brood size = 4, n = 32, P = 0.849; brood size = 5, n = 15, P = 0.753; brood size = 6, n = 11, P = 0.794).


Offspring sex ratio in the Penduline Tit does not depend on which parent provides brood care, is not repeatable between broods of individual males or females, and does not deviate from parity considering the population as a whole. It also does not deviate from the binomial distribution, which suggests that there is no bias within broods towards male- or female-only offspring. Palmer (2000) and Ewen et al. (2004) criticize the reported deviations from a 1:1 primary sex ratio and the evidence that birds are able to modify their sex ratio in an adaptive manner (but see Hasselquist and Kempenaers 2002). However, Cassey et al. (2006) argue that an overall trend in facultative adjustment of offspring sex ratio is weak but significant using the same data as Ewen et al. (2004). Several examples of adaptive offspring sex-ratio adjustment in birds have been reported (e.g., Kestrel Falco tinnunculus, Dijkstra et al. 1990; Seychelles Warbler Acrocephalus sechellensis, Komdeur et al. 1997; and Blue Tit Cyanistes caeruleus, Sheldon et al. 1999; Korsten et al. 2006). Since these taxonomically diverse bird species appear to adjust offspring sex ratios, it seems unlikely that Penduline Tits are genetically or physiologically unable to do so. However, Griffin et al. (2005) suggested that variation in the occurrence of sex-ratio adjustment might be explained by the strength of selection for such adjustment, which differs across species. In the Penduline Tit, there may not be a clear fitness benefit of offspring sex-ratio adjustment. There is no clear sexual size dimorphism and thus the costs of raising male offspring are unlikely to be very different from that of raising female offspring. Furthermore, not only males but also females may obtain several mates within a given season (Szentirmai et al. 2007). So the reproductive success of male and female parent not only increases with the number of females their sons will mate with, but also with the rate of polygamy of their daughters. Similarly, if attractiveness would be heritable, this may involve a fitness advantage for both male and female offspring. Selection for adjustment of offspring sex ratio may therefore be weak in this species.

Our study also shows that, using data from 3 years, there is currently no evidence for a facultative adjustment of offspring sex ratio in Penduline Tits. This means that even if under certain conditions there may be a bias in offspring sex ratio, the frequency and robustness of such a bias in this species is likely to be low. This may have implications for the way we study sex allocation in vertebrates: in order to understand the evolution of adaptive sex-ratio modification, an unbiased literature with repeated studies, between years and within years between given individuals, is of primary importance. In this study, we show that the offspring sex ratio in successive broods of a given individual male or female is not repeatable, and that the distribution of males and females within broods is not different from the binomial distribution. These notions suggest that overall individual quality or individual traits such as plumage coloration or condition may not play an important role in the determination of an offspring’s sex. This is in line with most previous studies investigating repeatability of sex ratio within years (e.g., Leech et al. 2001; Westneat et al. 2002; Dietrich-Bischoff et al. 2006), although here contrasting results have also been reported (see, e.g., Whittingham et al. 2005). The selection to produce more sons when mated to an attractive male, for example, may be weak and counteracted by increased costs of producing attractive males or costs of manipulation itself (Fawcett et al. 2007). Rather, ecological factors such as temperature (Badyaev et al. 2003), territory quality and/or breeding opportunities may be paramount (Komdeur et al. 1997; Hipkiss and Hornfeldt 2004). For the latter, comparing different populations of the Penduline Tit may provide new insight, as at different sites across Eurasia, habitat quality, and in particular, breeding opportunities are likely to vary widely (D.M. Brinkhuizen, R.E. van Dijk, T. Székely, J. Komdeur, unpublished data). Recently, Griffin et al. (2005) showed that cooperative breeding species in general adaptively biased sex-ratios towards the helping sex, depending on the benefits that can be gained from having helpers around. To build on the present study, it would be of particular interest to investigate sex-ratios in the Cape Penduline Tit Anthoscopus minutus. This species is assumed to be closely related to the Eurasian Penduline Tit, yet exhibits facultative cooperative breeding (Dean 2005), in sharp contrast to the breeding system of the species investigated in the present study. This may shed further light on the link between the evolution of sex-ratio adjustment and cooperation (Griffin et al. 2005).


Das Geschlechterverhältnis bei der sequentiell polygamen Beutelmeise Remiz pendulinus

Trotz einer steigenden Anzahl von Studien über die Steuerbarkeit von Geschlechterverhältnissen bei Vögeln und Säugetieren, deren Geschlecht chromosomal bestimmt wird, weichen die Ergebnisse und Schlussfolgerungen unterschiedlicher Studien und Arten voneinander ab. In unserer Studie untersuchten wir über einen Zeitraum von drei Jahren das primäre und sekundäre Geschlechterverhältnis der Beutelmeise (Remiz pendulinus), eines kleinen eurasischen Singvogels. Diese Art ist gekennzeichnet durch ein einzigartiges Paarungssystem, bei dem entweder Männchen, Weibchen oder beide Eltern während der Eiablage den Partner und das Nest verlassen, um eine neue Brut zu beginnen. Dieses Verhalten gab uns die Möglichkeit zu untersuchen, (1) ob eine Beziehung zwischen Art der Brutfürsorge und Geschlechterverhältnis besteht, d.h. ob Brutfürsorge allein durch das Männchen, Weibchen oder Verlassen durch beide Eltern in einer Beziehung zum Geschlechterverhältnis des Nachwuchs stehen, und (2) ob die Geschlechterverhältnisse des Nachwuchses einer bestimmten Beutelmeise konsistent in verschiedenen Bruten sind. Unsere Studie fand keine Hinweise auf eine Steuerbarkeit des Geschlechterverhältnisses durch die Eltern in der Beutelmeise. Nach der Geschlechtbestimmung von 497 Jungvögeln in 176 Bruten durch die Verwendung von molekularen Markern konnten wir zeigen (1) dass das Geschlechterverhältnis nicht von der Art der Brutfürsorge abhängt und, (2) dass das Geschlechterverhältnis keine Konsistenz zwischen verschiedenen Bruten einer Beutelmeise zeigt. Weder primäres noch sekundäres Geschlechterverhältnis zeigten eine Abweichung von einem ausgeglichenen Geschlechtsverhältnis (Männchenanteil (Mittelwert ± Standardabweichung), primär: 54 ± 6%, sekundär: 50 ± 3%). Wir argumentieren, das eher ökologische und phänotypische Faktoren anstelle von individuellen elterlichen Merkmalen für das Geschlechterverhältnis beim Nachwuchs verantwortlich sind.


We would like to thank the people that assisted in the field work: Maarten Bleeker, Dušan Brinkhuizen, Kenneth Hayes, Péter Horváth, Arno wa Kang’eri, Sjouke Kingma, Otília Menyhárt and Lídia Mészáros. We also acknowledge Kiskunság National Park and Szegedfish Ltd for permission to carry out our fieldwork at Fehértó. The manuscript benefited from comments on earlier drafts by Tim Fawcett, Thomas Friedl, and one anonymous reviewer. The research leading to these results has received funding from the European Community’s Sixth Framework Programme (FP6/2002–2006) under contract no. 28696. Further financial support was provided by a University Studentship of the University of Bath to R.E.v.D., and grants from the Hungarian National Science Foundation (OTKA T031706, T043390) and a Royal Society Joint Project grant (15056) to T.S. T.S. was also supported by The Leverhulme Trust (RF/2/RFG/2005/0279) and a HRDY Visiting Fellowship of Harvard University. The fieldwork carried out for this study complies with the Hungarian law.

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© Dt. Ornithologen-Gesellschaft e.V. 2008