Uropygial gland volatiles facilitate species recognition between two sympatric sibling bird species

Behavioral Ecology The official journal of the ISBE International Society for Behavioral Ecology Behavioral Ecology (2013), 24(6), 1271–1278. doi:10.1093/beheco/art068 Yao-Hua Zhang, Yu-Feng Du, and Jian-Xu Zhang The State Key Laboratory of Integrated Management and Research of Insect and Rodent Pests in Agricultures, Institute of Zoology, Chinese Academy of Sciences, Beichenxi Road 1-5, Beijing 100101, China Received 3 April 2013; revised 8 June 2013; accepted 10 June 2013; Advance Access publication 13 August 2013 Visual and acoustic cues in birds have been well documented to play a role in species recognition between closely related bird species. Here, we aimed to test whether chemical cues also play a role in avian species recognition between 2 sympatric sibling species of waxwings, Bombycilla garrulus and Bombycilla japonica. Using gas chromatography–mass spectrometry, we characterized from uropygial gland secretions 38 compounds that were quantitatively divergent between species and exhibited sex differences within species. Nine major compounds, including 6 linear alkanols and 3 carboxylic acids, which accounted for more than 85% of all compounds were used for simulation of the scents. Female B. garrulus exhibited a striking preference for their mirror images scented with either conspecific body odor or its synthetic analogs and avoided the scents of the sibling species B. japonica in a Y-maze olfactometer. Our results suggest that the volatile components of uropygial gland secretions have diverged in composition and these differences contribute to species recognition between sympatric sibling bird species and subsequently affect the likehood of speciation. Key words: avian olfaction, odorant signals, speciation, waxwings. Introduction Sympatric sibling species are important models to study speciation, as species recognition is important in premating isolation and maintaining species boundaries in animals (Mallet 1995; Panhuis et al. 2001; M’Gonigle et al. 2012). The divergent signaling traits, which contribute to species recognition and premating isolation between sibling species, are presumed to have promoted speciation when the most recent ancestor species split into separate species (Panhuis et al. 2001). The variation in chemical signals or pheromones may drive species divergence and play an important part in premating isolation among species (Löfstedt et al. 1986; Baker 2002). The importance of olfaction in conspecific recognition has been shown in closely related sympatric species of fish (McLennan and Ryan 1999; Rafferty and Boughman 2006), salamanders (Dawley 1984), lizards (Barbosa et al. 2006), rodents (Todrank and Heth 2003), and many insects (Singer 1998). Investigations of chemical signals involved in speciation of moths and fruit flies suggest that small changes in ratios or types of isomers of pheromones can create Address correspondence to J.-X. Zhang. E-mail: . © The Author 2013. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please e-mail: a reproductive isolation barrier between closely related species (Silverstein et al. 1966; Löfstedt 1993; Wyatt 2003; Bengtsson and Löfstedt 2007). In contrast to other animals, studies of signals in avian communication have been extensively focused on vocal and visual cues but not chemical cues (Hagelin et al. 2003; Wyatt 2003; Campagna et al. 2012). Because postzygotic isolation in birds is not well developed, speciation mainly relies on prezygotic isolation based on vocal and visual cues to prevent gene flow (Price and Bouvier 2002; Edwards et al. 2005; Uy et al. 2009; Clark 2011; Pfennig and Hurlbert 2012). Interspecific differences in visual and auditory signals (such as songs, plumage coloration, and ultraviolet plumage reflectance) are used by sympatric closely related birds in species recognition for mate choice or sexual competition (Andersson et al. 1998; Panhuis et al. 2001; Boughman 2002; Bleiweiss 2004; Matyjasiak 2005; Uy et al. 2009). Chemical cues in birds are now known to play a greater role in a variety of behavioral situations than was previously recognized, for example, in individual and partner recognition (De Leon et al. 2003; Hagelin et al. 2003; Bonadonna and Nevitt 2004; Whittaker et al. 2011), nest and kin recognition (Bonadonna and Mardon 2010; Caspers and Krause 2011; Coffin et al. 2011; Bonadonna and Sanz-Aguilar 2012; Krause et al. 2012), predator assessment Uropygial gland volatiles facilitate species recognition between two sympatric sibling bird species Editor’s choice Original Article Behavioral Ecology 1272 Materials and Methods Study species Sixteen male and 16 female B. garrulus and 8 male and 8 female B. japonica were captured in the winter of 2010 in Hebei province, China. Birds were randomly paired with opposite-sex conspecifics, and each pair was kept in a wire cage (47.5 × 33 × 32.5 cm). Pairs were exposed to a photoperiod of 13:11 h light:dark and a temperature of 24 ± 3 °C. Tap water and dry mash of feedstuff for chicken supplemented with mealworms and berries were provided ad libitum. The experiments were performed from April to July 2011 during the breeding season. Because waxwings are sexually monomorphic, each bird was sexed using DNA extracted from blood. The primers 2550F 5′-GTTACTGATTCGTCTACGAGA-3′ and 2718R 5′-ATTGAAATGATCCAGTGCTTG-3′ were used to amplify the CHD gene, which produces a single Z band in males, and Z and W bands in females (Fridolfsson and Ellegren 1999). Chemical analysis The UPGS and feather collected from live birds were extracted with dichloromethane, and then analyzed by GC–MS, as previously used in budgerigars (Zhang, Wei, et al. 2010). In brief, we gently sanitized both lobes of the gland with 75% ethanol swabs and squeezed the area around the gland to collect the UPGS and used a pair of scissors to cut off the feather tips from back, belly, and wings; the UPGS and feather samples were stored in vials at −20 °C till use (Zhang, Wei, et al. 2010). Dichloromethane (purity >99.5%; Dikma Technology, Inc.) was added into the vials at a proportion of 1-mg UPGS or 2-mg feather in 40 μL solvent, and the samples were incubated at 0 °C for 12 h. Chemical analysis was carried out with a 6890 Agilent gas chromatograph coupled to a 5973 mass spectrometer and the MS Library NIST2002 (Agilent Technologies, Inc.). The GC had an HP5-MS column (30 m × 0.25 mm internal diameter × 0.25 μm film thickness, Agilent Technologies, Inc.), carrier gas helium at 1.0 mL/min, and injector set at 280 °C. The oven was programmed at 5 °C/min from 70 to 280 °C. MS was in the electron impact mode 70 eV, and the transfer line was set at 280 °C (Zhang, Wei, et al. 2010; Zhang 2011). Three microliters of the extract were injected in the splitless mode. We calculated relative abundance for each volatile of each bird as a GC peak area percentage of the sum of the 3 (...truncated)