zy zyxwvu zyxwvutsrqpon zyxwvutsrqp Hereditas 117: 103-108 (1992) DNA fingerprinting reveals multiple paternity in families of Great and Blue Tits (Parus major and P. caeruleus) ANNICA GULLBERG, HAKAN TEGELSTROM and HANS P. GELTER Department of Genetics, Uppsala University, Uppsala, Sweden zyxwv zyxwv zyxw zyxwvuts zyxw GULLBERG, A., TEGELSTROM, H. and GELTER,H. P. 1992. DNA fingerprinting reveals multiple paternity in families of Great and Blue Tits (Parus major and P. caeruleus). - Hereditas 117: 103-108. Lund, Sweden. ISSN 0018-0661. Received December 15, 1991. Accepted January 23, 1992 Paternity of nestlings in the great tit (Parus major) and blue tit (Parus caeruleus) was studied using DNA fingerprinting. Multiple paternity was found in five out of ten great tit families and two out of seven blue tit families. Among the great and blue tit families 7 out of 47 (15 %) and 3 out of 51 (6 %) of the nestlings, respectively, were the result of extra-pair matings. Thus, extra-pair fertilization was proven not only in the blue tit but also in the great tit, a species regarded to be strictly monogamous. In no case was the whole brood sired by an extra-pair male and no case of intraspecific brood parasitism was detected. Annica Gullberg, Department of Genetics, Uppsala Uniuersity, Box 7003, S - 750 07 Uppsala, Sweden Unique patterns, DNA fingerprints, can be obtained after hybridization with probes for hypervariable minisatellite loci that are dispersed in the genome (JEFFREYS et al. 1985), making it possible to analyse the parentage of individuals in a population (BURKE 1989). In birds, extra-pair sexual activities are not rare and may be more common than expected from observations alone (for reviews, see MCKINNEYet al. 1984; BIRKHEAD 1987). Several studies using DNA fingerprinting have verified extra-pair parentage in different bird species (WETTONet al. 1987; BURKEet al. 1989; BIRKHEAD et al. 1990; GIBBS et al. 1990; RABENOLD et al. 1990; WESTNEAT1990), but also absence of illegitimate nestlings in the monogamous willow warbler and the polygynous wood warbler (GYLLENSTEN et al. 1990). The possible occurrence of successful extra-pair copulations (EPC), resulting in extra-pair fertilization (EPF) may severely bias estimates of lifetime reproductive success which is of great importance for the evaluation of mating strategies and social organization. The great tit (Parus major) and the blue tit ( P . caeruleus) are secondary hole nesters breeding in similar woodland habitats. They readily accept nestboxes, which make them easy to census and study. After nesting, the breeding pair abandon their territory but it is usually reoccupied early in the following spring. Pair-bonds are formed prior to or early in the breeding season (HARVEY et al. 1979). The great tit breeds asynchronously and males defend large territories and guard their mates (BJORKLUND and WESTMAN 1986; BJORKLUND et al. 1992). Both species are con- sidered monogamous, although the blue tit occasionally becomes polygynous ( DHONDT1987). We have used DNA fingerprinting to investigate the possible occurrence of extra-pair paternity or intraspecific brood parasitism in the apparently monogamous great tit and in the closely related blue tit. Materials and methods During spring 1988 ten great tit and seven blue tit families were sampled for DNA fingerprinting analysis from a nestbox area a few km south of Uppsala, Sweden. Nestboxes were set out before the breeding season in 1987 in a mixed coniferous and deciduous forest. Between 50 and 1 0 0 ~ 1of blood from 67 great tits and 65 blue tits (the two attending parents and nestlings) were collected in capillary tubes from a short cut with a scalpel on the middle of the tarsus. The pair of birds attending the nestbox and feeding the nestlings was regarded as the putative parents. Blood was transferred to polypropylene tubes with 1 0 0 ~ 1SSC buffer (0.15 M NaCI, 0.15 rnM trisodium citrate, 0.5 mM EDTA, pH 7.0) and stored at - 70°C. The number of eggs, nestlings (Table 1) and hatching dates (data not shown) were recorded for each family. All nestlings could not be analysed because they had already left the nest at the time of blood sampling. 104 zyxwvutsr zyxwvutsrqponml zyxwvutsrqpo zyx zyxwvutsrqponm A GCLLBERG ET 4~ Heredtior I I7 (1992) Tabk I Family number. the number of eggs (unhatched in brackets). the number of nestlings analysed with DNA fingerprinting dnd the number of EPF-offspnng found in each family of great ( A ) and blue ( 9 ) ti!$ A Great t i t zyxwvutsrqponmlk zyxwvutsrqponm zyxwvutsrqp Family II 16 39 56 72 91 98 105 I08 I18 B Blue Family 10 15 in 55 70 I01 103 VOCELSTEIN 1983). Prehybridization and hybridization were performed according to GEORCES et al. (1988). Membranes were washed 2 x 15 min in 1.5 x SSC, 0.1 %) SDS at room temperature, 2 x 15 min in 1.5 x SSC, 0.1 % SDS at 60°C and finally 10 min in 1 x SSC at room temperature and autoradiographed at -70°C for 1-6 days using Kodak X-omat AR and intensifying screens. Most membranes were subjected to different exposure times to make visible bands of different intensities. The DNA probes were removed from membranes by washing in 0.4 M NaOH and 0.2 M Tris-HC1, pH 7.5. checked for remaining radioactivity, and then rehybridized with the next probe. Comparisons of the same membrane hybridized with both probes show that they detect different minisatellite alleles and, hence, results from the two probes can be combined as independent. No of eggs Analysed no. of offspring No. of EPFoffspring 6(l) 5 4 3 6 7 3 1 1 2 7 4( 1) 7 7 9 0 0 2 0 7 1 6 71) 8 3 1 6 0 8 0 N o of eggs Analysed no of offspring N o of EPFoffspring 7 4 7 5 I1 I1 6 0 2 tit 7 h 9 90) 13 11 6 0 0 0 I 0 Genomic DNA was extracted by addition of 2.5 ml SET-buffer (0.15 M NaCI. 0.05 M Tris-HCI. 1 mM EDTA, pH 8.0). 50 pl of 25% SDS (wiv), and 80 pI of proteinase K (10 mglml). The tubes were gently shaken for 4 h at 3 7 T . and DNA was purified by two extractions with phenoljchloroform and two with chloroform. DNA was precipitated with ethanol and dissolved in 0.4-1.5 ml 0.01 M ’Tris-HCI. pH 8.0 for at least 24 h. DNA (8-10 pcg) was digested with 30 units of Hae I11 for 4 h at 37.C. extracted once with phenoljchloroform, once with chloroform, and precipitated with ethanol. The digested DNA was dissolved in 25 pl 0.01 M TrisliC1. pH 8.0. DNA fragments were separated in I5 x 26 cm 0.8 YOagarose gels for 24--28 h at 1.7 V/ cm and transferred to Pall Biodyne A transfer membranes by vacuum blotting. The insert of the human minisatellite clone 33.15 ( JEFFRFYS et al. 1985) and a Cla I/Bsa 1-785 basepair fragment from wild type M13 (VASSARTet al. 1987) were isolated by preparative restriction enzyme digestion and electrophoresis in low melting temperature agarose. The probe DNA was purified from the agarose using Gene Clean (Bio 101). 50--75 ng of probe DNA were 32P-dCTPlabelled by the random primer method (FEINBERG and Results zyx The mean number of analysed bands ( > 3 - 4 kb) per individual (n) was 15.2 i 8.0 SD ( M i 3 probe) and 16.2 f 5.6 SD (33.15 probe) for the great tit and 14 3 f 6.6 SD (M13 probe) and 15.1 & 3.1 SD (33.15 probe) for the blue tit. The average bandsharing (x) between unrelated individuals (breeding pairs) calculated according to WETTONet al. (1987) was 0.18 (MI3 probe) and 0.25 (33.15 probe) for the great tit and 0.17 (M13 probe) and 0.14 (33.15 probe) for the blue tit. The degree of bandsharing was similar to that found in other bird species for the 33.15 probe; range 0.17-0.27 (BURKE and BRUFORD1987; BURKEet al. 1989; BIRKHEAD et al. 1990: GIBESet al. 1990; RABENOLD et al. 1990; WESTNEAT1990). The estimated mean population allele frequency (4) was 0.11 for the great tit and 0.08 for the blue tit, assuming an independent segregation of identified bands (q = 1 - (1 - x)”’, JEFFREYS et al. 1985). The probability that two randomly selected individuals will show the same band pattern with both probes combined (M 13 and ((1 - 2 x + 33.15) was 7 x lo-” and 6 x 2x’)” ’, JEFFREYS and MORTON1987) for the great and blue tit, respectively. Among the great tit families 13 out of 47 of nestlings, and among the blue tit families 7 out of 51 of nestlings showed at least one band not present in either parent (Fig. 1). These mismatched bands can either be explained by the high mutation rate characterizing minisatellite loci (JEFFREYS et al. 1988) or by extra-pair parentage. If mismatched zyx zyxwvutsrqponm zyxwvutsrqponml zyxwvutsrqponm Hereditas 117 (19YZ) MULTIPLE PATERNITY IN GREAT AND BLUE TITS 105 zyxwvutsrqp zyxwvu Fig. 1. Examples of DNA-fingerprints (probe 33.15) showing multiple paternity among offspring of the great tit (A) and blue tit (B). Bands not present in either putative parent are indicated by an arrow. bands were due to mutation alone they would be randomly distributed among the nestling. The probability that an offspring will have b mismatched bands can be obtained from the Poisson distribution e--mmb/b!,where m is the mean number of mismatched bands per individual (BURKE and BRUFCJRD1987). This analysis (left half of Table 2A) shows that the mismatched bands in seven great tit nestlings cannot be explained by mutation alone and that these nestlings most probably were the result of extra-pair parentage. Among the remaining six nestlings showing mismatched bands, all but one (1 1-1, Table 2A) could be assigned to the expected father (see below). Offspring 11-1 has fewer bands than the other nestling in the family due to a lower amount of DNA on the gel. Some of the less intense paternal bands may have remained undetected in offspring 11-1, which may explain why there were fewer bands than expected. Mismatched bands in four of the blue tit nestlings (Table 2B) could be explained by mutational events, which also is supported by an analysis of the number of paternal bands (see below). When the EPF-nestlings have been excluded, the mutation rate can be estimated by comparing the remaining mismatching bands with the total number of bands in the non-EPF nestlings (RABENOLDet al. 1990). The mutation rates were 8 x and 3 x in the great tit and blue tit respectively, which is within the range found for other bird species (2 x - 11 x lop3, BURKEand BRUFORD1987; WESTNEAT 1990). Assuming a high level of heterozygosity, the offspring should show about half of the bands specific for the father and half of the bands specific for the mother. If a nestling only has a few bands in common with a putative parent, this individual probably is not the real parent. Bands that are shared between the parents cannot be used in a parentage analysis and are neglected. Assuming independent segregation of bands, the probability that an offspring will have the observed number or less of the paternal specific bands can be found by summing up the relevant terms of the binomial distribution (!)PL(I where N is the number of bands specific for the father, k is the number of these bands found in the zyxwvutsrqponml zyxwvutsrqpo zyxwvutsrqpon zyxwvuts zyxwvu zyxwvutsrqp zyxwvu zyxwvu zyxwv Trrhir 2. Anal)si$ o f whether mrbmatched bands in a nestling are due to mutation or to extra-pair fertilization. The left part of the table 5hows the number of mismatched bands per nestling (among the nestlings showing mismatched bands) and the estimated probability of obtaining h i t number hq chance for the greal tit ( A ) and the blue tit (B).The right part of the table shows the number of bands \pecific for the putative father. the number of these bands expected to be found in each nestling (50%) and the number of bands actuallg found. The probahilip balue gives the estimated probability that a nestling will have the observed number or less of the paternal specific hands just h! chance I f this probability is lower than 50'0. the oRsprinp is classified as an EPF-nestling .4. Grsat t i t Fami -!I nestling Mismatched band\ Probability Male specific bands Expected no. of bands Observed no. of bands Probability 38 38 4X 48 19 19.0 I? 3 28 0 02 33x10 0 90 4 2 x 10W" 0 92 19 19 21 30 30 i- .tinil) ne5iling hlicmatched bmJ\ Pro babi Ii t y 19.0 24.0 24.0 9.5 9.5 9.5 11.0 11.0 11.0 2 12 1 I 4 13 7 15.0 '0 2' 15.0 11.5 14 6 Male ~peciti~: bands Expected no. of bands Ohserved no. of bands 15.5 0 lj5 15.5 12.0 20 0 12.0 14.0 14.0 ofspring. and p is the probability that a band will be transferred from parent to offspring. If the level ol' hctcrorygosity is high, p will be close to 0.5. Should the level of heterozygosity be lower than assumcd. using p = 0.5 will overestimate the numhcr of nestlings found to be the oft'spring of the putativc parent and underestimate the level of EPF. The results of the paternity analyses are shown in the right half of Table 2. Among the thirteen great tit offspring showing mismatched hands. tight showed a significantly (on the 5 % letel) lower number of bands than expected from thc putativc father. All of these (except offspring 1 1 - 1 ) were the same as identified by the mutation analysis above confirming that seven great tit nestlings in five families werc the result of EPF. The three blue tit offspring that could not be explained bq mutational events also had a significant difference between expected and observed numbers of paternal bands, confirming their EPF origin (Table 3B). 38x10 38x10 22x10 0 86 6 0 x 10 0 98 0 43 73x10 ' Probability 4 1 * 10 0 96 4 7 x 10 0.58 0.27 1.5 x 10 0.29 'I1 zyxwvu 12 10 2 'I' ~h In 5 out of 10 ( S O 0 / , ) of the great tit families and in 2 out of 7 (29%) blue tit families at least one nestling was not the descendant of the expected father. Altogether 7 out of 47 (15%) great tit and 3 out of 51 ( 6 % ) blue tit nestlings had an extrapair male as a father. The 95% confidence intervals extend from 7 to 28% and 1 to 17%, respectively. In no case was the whole brood sired by an EPF male. Two EPF-nestlings in three families (15, 39, and 91) shared 3, 7, and 5 of the mismatched bands, respectively, indicating that the EPF-nestlings in each family might have had the same father. The higher EPF frequency of the great tit compared with that of the blue tit was not significant (Fisher's exact probability test, p = 0.27). An analogous analysis of maternal bands showed that in none of the nestlings did the number of maternal-specific bands deviate significantly from the expected (data not shown). Thus, no case of intraspecific brood parasitism was detected. zyx zyxwvutsrqponm zyxwvutsrqponmlkj zy MULTIPLE PATERNITY IN GREAT AND BLUE TITS Heremius I 1 7 (1992) 107 zyxwvu The average nearest neighbour distance between the nestboxes of the great and the blue tit were 116 m rf: 52 SD and 94 m 50 SD, respectively. The average distance for the “faithful” great tit families was 136 m 62 SD compared to 97 m f 35 SD for the EPF families but the difference was not significant (Mann-Whitney, u = 18). Nor did the hatching date differ significantly between the EPF and “faithful” families (Mann-Whitney, u = 8.5). Discussion The present report, using DNA fingerprinting, shows that extra-pair fertilization occurs in both great and blue tits. The proportion of offspring resulting from extra-pair paternity in our particular samples of the great tit (15 %) and the blue tit (6%) is within the range found in other avian species (0-40%, BIRKHEADand MOLLER1991). In a recent study using DNA fingerprinting, DHONT(pers. commun.) found a similar EPF frequency ( 1 1 %) in a population of blue tits outside Antwerp (Belgium). The percentage of extrapair paternity among broods of great tits is 50% but because of the small sample size the confidence interval (95%) extends from 19 to 81 %. This interval overlaps the range found in other species (range 0-48%, BIRKHEADand MOLLER 1991). Although lone female great tits appear to reject EPC attempts as actively as accompanied females, lone females encounter soliciting males at a high rate (BJORKLUND et al. 1992). As demographic factors (such as age structure and density of a population) probably are important in determining the occurrence and frequency of EPF, the extent of EPF may vary between populations. Our particular great and blue tit populations were newly established when sampled and this might have had a significant effect on their demography and the occurrence of EPF. To distinguish between different factors affecting the frequency of extra-pair fertilization in the great and blue tit, paternity analyses must be combined with detailed behavioural observations and demographic data throughout the breeding season. This kind of combined studies will make it possible to investigate when and why extra-pair fertilization occurs. Is EPF a result of rapid mate switching, has the nest-owner been cheated by a male seeking extra-pair copulation, or has the female actively searched copulations with neighbouring males? Acknowledgements.-We would like to thank Per-Ivan Wyoni for suggesting a bionomial test of paternity, and for writing a computer program for the calculations. We are grateful to AIec J. Jeffreys for providing the minisatellite probes, and to Karl Fredga and Peter 0. Dunn for reading previous drafts of this report. The Swedish Natural Science Research Council and the Erik PhilipSorensen Foundation supported this research. zyxwv zyxw zyx zyx zyxwvu References BIRKHEAD, T. R. 1987. Sperm competition in birds. - Trends Ecol. Evol. 2: 268-272 BIRKHEAD, T. R. and MOLLER,A. P. 1991. Sperm Competition in Birds: Evolutionary Causes and Consequences. - Academic Press, London BIRKHEAD, T. 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