© 2016. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2016) 219, 1961-1964 doi:10.1242/jeb.138263 SHORT COMMUNICATION Incubation temperature causes skewed sex ratios in a precocial bird Sarah E. DuRant1,*, William A. Hopkins2, Amanda W. Carter2, Laila T. Kirkpatrick3, Kristin J. Navara4 and Dana M. Hawley3 ABSTRACT In birds, females are the heterogametic sex and there is evidence Many animals with genetic sex determination are nonetheless demonstrating that female birds are capable of manipulating primary capable of manipulating sex ratios via behavioral and physiological sex ratios (sex ratio at fertilization) prior to fertilization (Krackow, means, which can sometimes result in fitness benefits to the parent. 1995; Navara, 2013). However, birds exhibit considerable parental Sex ratio manipulation in birds is not widely documented, and care post-oviposition and post-hatching, with both sexes revealing the mechanisms for altered sex ratios in vertebrates contributing to care in many species. Thus, opportunities exist for remains a compelling area of research. Incubation temperature is a parents to manipulate sex ratios after egg laying (i.e. secondary sex key component of the developmental environment for birds, but ratios). For example, incubation temperature could influence despite its well-documented effects on offspring phenotype it has secondary sex ratios of birds via differential embryonic mortality rarely been considered as a factor in avian sex ratios. Using between the sexes (Eiby et al., 2008; but see Collins et al., 2013). ecologically relevant manipulations of incubation temperature within Avian temperature-dependent sex-biased embryonic mortality the range 35.0–37.0°C, we found greater mortality of female embryos (TDSEM) was first documented in a Megapode species (Eiby Alectura lathami during incubation than males regardless of incubation temperature, et al., 2008), the Australian brush turkey ( ). Unlike and evidence that more female than male embryos die at the lowest most other birds, which use contact incubation (i.e. transfer of heat incubation temperature (35.0°C). Our findings in conjunction with from parents to eggs through physical contact), Megapodes use previous work in brush turkeys suggest incubation temperature is an environmental heat from decomposing plant matter to incubate their important determinant of avian secondary sex ratios that requires eggs, and thus this avian group may be unique in showing TDSEM. additional study, and should be considered when estimating the Although not observed in an ecological context, three more recent impact of climate change on avian populations. studies in commercial poultry have demonstrated altered secondary sex ratios under differing incubation conditions (Tzschentke and KEY WORDS: Parental effects, Secondary sex ratio, Wood duck, Halle, 2009; Yilmaz et al., 2011; Piestun et al., 2013), and faster Incubation growth of male chicks post-hatch under conditions that produced more males (Tzschentke and Halle, 2009). However, these studies INTRODUCTION did not identify whether skewed sex ratios in poultry were the result Many vertebrates, including those with genetic sex determination, of TDSEM. Thus, it remains unknown whether the occurrence of can manipulate offspring sex ratios at various stages of development TDSEM is unique to Megapodes, and whether relatively large (Krackow, 1995; Pike and Petrie, 2003; Uller et al., 2007), including differences in incubation temperature are required to elicit sex- prior to fertilization, throughout embryonic development, and biased embryonic mortality in birds. post-parturition or post-hatching (Pike and Petrie, 2003). In some Unlike Megapodes, wood ducks (Aix sponsa) use contact cases, manipulation of sex ratios could be adaptive by producing incubation and nest in cavities, which is thought to be associated more males or females when conditions are more favorable for one with relatively low variation in incubation temperature among nests sex over the other (Charnov and Bull, 1977; Warner and Shine, compared with Megapodes (reviewed in DuRant et al., 2013a). 2008a,b; Pen et al., 2010). For instance, in parrot finches (Erythrura However, substantial evidence indicates that even small variation in trichroa), mothers produce more sons under environmental average incubation temperature (∼1–2°C) can alter a suite of conditions where daughters, but not sons, have high juvenile phenotypic traits (DuRant et al., 2013a,b) including evidence of mortality (Pryke and Rollins, 2012). Although the mechanisms skewed secondary sex ratios (this study). Specifically, more male underlying deviations from balanced sex ratios are well studied in wood duck hatchlings are produced at the lowest incubation some vertebrate groups (e.g. reptiles), the mechanisms producing temperature (35°C) compared with higher temperatures (Fig. 1). skewed ratios in birds, which exhibit genetic sex determination, Based on this evidence, we determined the sex of the dead embryos remain largely unknown (Navara, 2013). to test whether TDSEM occurs in wood ducks, which, if present, could suggest that TDSEM occurs in other contact-incubating birds. 1Department of Integrative Biology, Oklahoma State University, Stillwater, Incubation temperatures used in this study represented the cooler, OK 74075, USA. 2Department of Fish and Wildlife Conservation, Virginia Tech, warmer and modal incubation temperature that a natural population 3 Blacksburg, VA 24061, USA. Department of Biological Sciences, Virginia Tech, of wood duck eggs experiences (Hepp et al., 2006). Blacksburg, VA 24061, USA. 4Poultry Science, University of Georgia, Athens, GA 30602, USA. MATERIALS AND METHODS *Author for correspondence ([email protected]) Study species S.E.D., 0000-0003-0724-0878 Wood ducks, Aix sponsa (Linneaus), are a wide-ranging and common bird in North America. In our population, females nest Received 28 January 2016; Accepted 27 April 2016 from February to June, receive no aid from males during incubation, Journal of Experimental Biology 1961 SHORT COMMUNICATION Journal of Experimental Biology (2016) 219, 1961-1964 doi:10.1242/jeb.138263 0.7 Males volumes, and amplified with 3 µl of template DNA in a DNA Females Engine Dyad Peltier Thermal Cycler (Bio-Rad, Hercules, CA, 0.6 USA) using an annealing temperature of 55°C. PCR products were separated using 3% agarose gels in SB buffer and visualized with 0.5 SYBR® Safe DNA gel stain (Invitrogen Life Technologies, Grand Island, NY, USA). 0.4 Statistical analyses 0.3 Statistical analyses were performed using JMP and SAS (SAS Institute, Carey, NC, USA) software, and statistical significance was recognized at α=0.05. To determine whether sex ratios of hatchlings 0.2 63 38 44 56 47 37 Proportion of individuals differed across incubation temperatures, we used logistic regression. We also determined whether embryonic mortality of the sexes 0.1 differed across incubation temperatures with a logistic regression model (JMP, SAS Institute) where mortality (survived or died) was 0 35.0°C 35.9°C 37.0°C the dependent variable and sex, temperature and their interaction were independent variables. We also tested for differences in Fig. 1. Percentage of male and female wood ducks that hatched at each embryonic mortality within incubation temperatures and within the incubation temperature. Numbers inside bars represent the number of males sexes using a series of logistic regressions. or females that hatched. RESULTS AND DISCUSSION and begin incubation after clutch completion (Hepp et al., 2006). We detected significant differences in secondary sex ratios of Clutch size varies from 8 to 16 eggs, and nest parasitism can result in ducklings (Fig. 1; χ2=6.9, P=0.03), and provide evidence that clutches of >25 eggs. Both the timing of nesting and clutch size can skewing of the sex ratio may occur through TDSEM. Embryonic influence temperatures that eggs experience (reviewed in DuRant mortality significantly differed between the sexes (Fig. 2: χ2=4.01, et al., 2013a). P=0.045), and this difference appeared to be present only at the lowest incubation temperature (Fig. 2; individual effect test for Egg incubation females at 35.0°C versus 35.9°C: P=0.047), but we were unable to We collected eggs from wood duck nests on the Savannah River Site detect a statistically significant interaction between incubation in Aiken, SC, USA, during the spring of 2011. We artificially temperature and sex on embryonic mortality (Fig. 2; temperature: incubated 380 eggs collected from 58 nests, which were evenly χ2=3.77, P=0.15; sex×temperature: χ2=4.23, P=0.12). However, distributed across incubation temperature treatments to account for individual logistic regression analyses of embryonic mortality of genetic and non-genetic parental effects. In many cases, eggs from a males and females within each temperature support TDSEM; single nest were produced by multiple females, because wood ducks significantly more females died than males at 35.0°C (χ2=3.96, exhibit intraspecific brood parasitism. Eggs were incubated at P=0.046), while a similar number of males and females died at 35.9 overall average temperatures of 35.0, 35.9 or 37.0°C in Grumbach and 37.0°C (P≥0.37 in both cases). This is the first evidence that incubators (BSS 160) at Virginia Tech. Temperatures in incubators incubation temperature can lead to skewed sex ratios through decreased twice daily by ∼3°C to mimic recesses taken by females to forage (temperature reductions during recesses in field, 2–6°C). Temperatures in incubators were verified continuously throughout 40 the study using i-button temperature loggers (model no. DS1923). Males Hatching success was 69–77% (hatching success in the wild is Females ∼80% on average; range, 37.5–100%; S. F. Hope, S.E.D. and 35 W.A.H., unpublished data) and did not differ among incubation temperatures (P=0.27). Further details on egg collection, incubation 30 and treatment assignment in 2011 have been published elsewhere (DuRant et al., 2013b). All procedures were approved by the 25 Virginia Tech Animal Care and Use Committee. 20 Sex identification For ducklings that hatched, we identified sex after they were 15 euthanized by asphyxiation with CO2.