2011 SOUTHEASTERN NATURALIST 10(2):365–370 Nestling Sex Ratios in Two Populations of Northern Mockingbirds Brett E. Schrand1,3, Christopher C. Stobart1,4, Dorothy B. Engle1, Rebecca B. Desjardins2, and George L. Farnsworth1,* Abstract - In birds, sex is determined by the allocation of sex chromosomes. We used molecular techniques to determine the sex of Mimus polyglottos (Northern Mockingbird) nestlings in Cincinnati, OH and Raleigh, NC. We found an overall male-biased sex ratio in the Cincinnati population and a female-biased sex ratio in the Raleigh population. In Cincinnati, the male-biased sex ratio was more pronounced early in the breeding season than later in the breeding season. Male nestlings were heavier than female nestlings and may require greater parental investment. In many avian species, female offspring are more likely to disperse, making them less likely to compete with parents and siblings for local resources in future seasons. This may explain why the Raleigh population with greater nesting population density overproduced female offspring. Introduction Sex in birds is determined by allocation of sex chromosomes (where males are homogametic and females heterogametic). Mendelian segregation during oogenesis suggests each fertilized egg should have the same probability of producing a male or female embryo. And from an evolutionary perspective, sex allocation theory predicts parents should invest equally in the production of male and female offspring (Fisher 1958). Thus, the sex ratio of a species in which males and females cost the same to raise should be unity. Roughly equal numbers of male and female nestlings have been observed in wild populations of some bird species such as Miliaria calandra L. (Corn Buntings; Hartley et al. 1999) and Emberiza citrinella L. (Yellowhammers; Pagliani et al. 1999). Here we present the first investigation of the nestling sex ratio of Mimus polyglottos L. (Northern Mockingbird). We expected to observe roughly equal numbers of male and female Mockingbird chicks. However, there may be situations in which it is adaptive to overproduce one sex. In some species, there may be differences in the cost of raising the two sexes that may alter the relative reproductive value of different sexes. In dimorphic species with one sex requiring more food to raise than the other, food availability may influence the likelihood of producing viable offspring. Myers (1978) proposed that in times of food limitation, the primary sex ratio should be biased toward the cheaper sex so as not to reduce fecundity, and provided an example with the sexually dimorphic chicks of Agelaius phoeniceus L. (Red-winged Blackbirds). Similarly, 1Department of Biology, Xavier University, Cincinnati, OH 45207-4331. 2North Carolina State Museum of Natural Sciences, Raleigh, NC 27601. 3Current address - Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, FL 33136. 4Current address - Department of Microbiology and Immunology, Vanderbilt University, Nashville, TN 37232-2363. *Corresponding author - Farnsworth@xavier.edu. 366 Southeastern Naturalist Vol. 10, No. 2 Wiebe and Bortolotti (1992) found biased hatchling sex ratios in Falco sparverius L. (American Kestrels) that they attributed to female manipulation of the primary sex ratio toward the cheaper sex during conditions of lower food availability. In species with a discrete breeding season, it may also be adaptive to vary sex ratio within a season. For example, early-season broods of Alauda arvensis L. (Skylarks) were found to be male-biased (Eraud et al. 2006). Male nestlings grew faster, suggesting an adaptive primary sex ratio based on greater availability of resources early in the breeding season. Another adaptive explanation for intra-seasonal variation in a species’ sex ratio can be due to differences in opportunities for breeding in the following year for the two sexes (Daan et al. 1996). For example, a female-biased sex ratio was observed in early-season broods in Sturnus unicolor Temminck (Spotless Starling; Cordero et al. 2001). Females from early broods were more likely to breed in the following breeding season than females from later broods, whereas the likelihood of males breeding in their first year was not sensitive to fledging date. However, these and other adaptive explanations for biased sex ratios have received criticism for potentially being premature (Ewen et al. 2004, Palmer 2000). Perhaps studies reporting biased sex ratios in birds are those rare studies that have observed such ratios by chance. With the ease of testing sex offered by molecular techniques, many investigators have begun testing sex ratios as additional projects attached to other studies. The combination of under-reporting non-significant results and easily accepting results that conform to adaptive explanations could lead to drawing generalizations that are not warranted. Recent attempts to replicate former studies that found biased sex ratios have failed to find similar results (e.g., see Johnson et al. 2005, Maddox and Weatherhead 2009). The mechanism that female birds might use to manipulate the sex of embryos is not fully understood (e.g., see Goelich et al. 2009, 2010), but empirical evidence that many species manipulate sex of embryos has been mounting from both field and laboratory studies (see Alonso-Alvarez 2006 for a review). We investigated nestling sex ratios (determined from feather DNA) of two populations of Northern Mockingbirds. One population, in Cincinnati, OH, is close to the northern limit of the species’ range. The other population, in Raleigh, NC, is closer to the center of the species range. The geographical and ecological differences between these two populations may influence the nestling sex ratio. For example, the number of Mockingbirds counted each year on the Breeding Bird Survey route closest to Cincinnati had an average 4.97 birds between 1966 and 2007, while the route closest to Raleigh had an average of 31.73 birds per year (Sauer et al. 2008). We also tested for changes in sex ratio within the breeding season in the Cincinnati population. Northern Mockingbirds are nonmigratory, seasonally-nesting passerine birds with open-cup nests and modest sexual dimorphism (adult males are approximately 10% larger by mass than adult females; Derrickson and Breitwisch 1992). Mockingbirds typically lay clutches of four eggs and delay the onset of incubation until the penultimate egg is laid, exhibiting synchronized hatching. Typically all eggs hatch on the same day and altricial chicks grow quickly, fledging by the 12th day post-hatch (Derrickson and Breitwisch 1992). 2011 B.E. Schrand, C.C. Stobart, D.B. Engle, R.B. Desjardins, and G.L. Farnsworth 367 Methods Our main study site was on and around the campus of Xavier University in an urban habitat in Cincinnati, OH (39°9'N 84°29'W). Feathers and mass were obtained from Northern Mockingbird chicks approximately 10 days after hatching during the breeding seasons (May to August) of 2005–2009 from the Cincinnati population. We also collected feathers from nestlings in Raleigh, NC(35°47'N 78°40'W) between April and July in 2008 and 2009. Feathers were collected from nests found opportunistically at various stages during the nesting cycle as part of another study examining extra-pair paternity. We therefore could not be sure of the entire history of the nest before discovery. This prevented us from testing sex ratio on a per-brood basis. Nevertheless, our null hypothesis was that each sampled nestling should have equal probability of being male or female if receptive ova were formed with a Z or W chromosome with equal and independent probability and any mortality between conception and fledging was not dependent on sex. DNA was extracted from quill ends of breast feathers using a 500 μL Epicentre® QuickExtract DNA Extraction Protocol. The resulting solution was stored at -20 °C until required for use. DNA was amplified by polymerase chain reaction (PCR) using Promega® PCR Master Mix. We used 5 μl of DNA in a 25-μl reaction containing 2 μM each of primers P2 and P8 (Griffiths et al. 1998), and 5 mM MgCl2. Following an initial hot start denaturing at 94 °C, the tubes underwent 30 cycles of 94 °C for 30 sec, 45 °C for 1 min, and 72 °C for 1 min. After the last cycle, the reaction was finished at 72 °C for 7 min, and then held at 4 °C until being stored at -20 °C. Reaction mixtures were analyzed using a 2.5% agarose gel post-stained with SYBR® Green 1 (Molecular Probes, Inc.). Each chick was sexed at least twice, and adults of known sex were used as positive controls in all amplifications. We used χ2 tests to examine the overall sex ratio in the two populations. We used a null-hypothesis that each chick sampled had an independent probability to be male of 0.50. We used logistic regression (R version 2.12) to test for a seasonal trend in sex ratios within samples from the Cincinnati population by coding each sample with a season day equal to the number of days after 1 May when each nestling was sampled. To examine whether it was more costly to raise a male chick versus a female chick, we compared the masses of chicks at the time of sample collection. We used a t-test to compare masses between males and females from the Cincinnati population. However, chicks from different broods were handled at somewhat different ages (8–11 days post-hatch) and had different numbers of nest-mates, so we also compared chicks within broods. For broods with both sexes represented, we looked at the heaviest and lightest chick within the brood. We used a G-test of independence to compare the number of males and females that were the heaviest chick in the brood and the number of males and females that were the lightest in the brood. Results From the Cincinnati population, we successfully amplified DNA from 145 chicks from 52 broods, yielding 87 males and 58 females. In four broods, only a single sample was collected either due to only one chick surviving or to only one chick caught from a recently fledged brood. From the Raleigh population, we 368 Southeastern Naturalist Vol. 10, No. 2 successfully amplified DNA from 37 chicks from 20 Broods yielding 26 females and 11 males. Eight broods provided only one sample each due to only one chick being caught or only one sample amplifying DNA. The DNA amplification and visualization required for sufficient sex discrimination was successful for every chick sampled in the Cincinnati population, but failed in 5 samples from the Raleigh population, including all 3 chicks from one brood. The Cincinnati population exhibited a male-biased sex ratio (χ2 1 = 5.8, P = 0.016). The Raleigh population exhibited a female-biased sex ratio (χ2 1 = 6.1, P = 0.014). There was a seasonal trend in sex ratio in the Cincinnati population (Table 1), with the probability of a chick being male influenced by season day of fledgling (logistic regression coefficient = -0.016 ± 0.007 SE, z = 2.42, P < 0.02). We recorded masses for 53 male chicks and 30 female chicks in Cincinnati. Male chicks were heavier than female chicks (males: 35.6g ± 6.5 [SD], females 32.7g ± 4.1; t81 = 2.22, P = 0.029). In 19 mixed-sex broods for which we had mass data composed of 34 males and 27 females, males were more frequently the heaviest chick and females were more frequently the lightest chicks (Gadj =5.69, P = 0.017). A male was heaviest in 14 broods, and a female was heaviest in 6 broods (in one brood a male chick and a female chick had the same mass). A male was lightest in 6 broods, and a female was lightest in 13 broods. Discussion In both populations, our null hypothesis that each chick reaching sampling age should be equally likely to be male or female was unlikely to be true. This could have been due to either unequal production of embryos of each sex and/or due to differential survival of embryos to 10 days post-hatch. It seems unlikely that differential survival would have led to a bias toward males in the early season in Cincinnati and a bias toward females in Raleigh. Our samples were not random samples of the populations due to our use of DNA from multiple nestlings within a brood. Thus, we could have measured bias due to individual females overproducing one sex and the differences we observed between populations (and time of season) were thus perhaps due to sampling artifacts produced by the small number of broods used. However, as an initial investigation of sex ratio in this species, our results suggest sex ratio of nestlings in Mockingbirds is not as simple as was expected. There may be adaptive explanations that should be investigated in future work. In the Cincinnati population, we observed more male nestlings than female nestlings, particularly early in the breeding season. We also found males were Table 1. Numbers of male and female chicks sampled from Cincinnati separated into early-season broods (fledged before 15 June) and late-season broods. Year Early season males:females (# of broods) Late season males:females (# of broods) 2005 5:5 (4) 8:2 (4) 2006 9:5 (5) 7:7 (4) 2007 9:5 (5) 14:20 (12) 2008 18:3 (9) 5:5 (3) 2009 11:5 (5) 1:1 (1) Total 52:23 (28) 35:35 (24) 2011 B.E. Schrand, C.C. Stobart, D.B. Engle, R.B. Desjardins, and G.L. Farnsworth 369 more likely to be the heaviest chicks in mixed-sex broods, suggesting that males require more parental investment to raise. This early-season bias toward the presumably costlier sex may be adaptive as a response to greater availability of resources early in the breeding season (Eraud et al. 2006). However, the seasonal bias could represent an adaptive response to males benefitting more than females from fledging earlier in the breeding season, similar to that found in Falco tinnunculus L. (Eurasian Kestrels; Dijkstra et al. 1990). Perhaps earlier-fledged males have a greater probability of surviving the winter and/or breeding in their first summer compared to males fledged later in the season. The local resource competition hypothesis suggests that when there is a difference in natal philopatry between sexes, future competition for resources between parents and the philopatric sex may reduce the residual reproductive value of the parents (Clark 1978). It may be adaptive to overproduce the dispersive sex when there is more local competition and overproduce the philopatric sex when there is less local competition. In most species of passerine birds, females are more likely to disperse than males (Clarke et al. 1997). If population density may be used as an indicator for local competition, it may be adaptive to overproduce females in the Raleigh population and males in the Cincinnati population. Data from the Breeding Bird Survey (see Sauer et al. 2008) suggests there is a higher population density of Northern Mockingbirds in Raleigh than in Cincinnati. Alternatively, the ratios we measured may not be adaptive responses to local or seasonal conditions. In these urban populations, pollution or contamination may have altered sex ratios in ways not related to selection pressures on nestling sex ratio. Experimental evidence has demonstrated that concentrations of sex hormones can influence primary sex ratio (Goelich et al. 2009). Exposure to environmental contaminants may alter primary sex ratio, as has been observed in Larus fuscus L. (Lesser Black-headed Gulls) exposed to environmental oganochlorines (Erikstad et al. 2009). This effect may be due to contaminants mimicking or disrupting sex hormones, altering stress hormone levels, or by affecting female body condition. 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