Study system and sample collection
A small number of breeding pairs of Oregon juncos (J. hyemalis thurberi
) colonized the University of California, San Diego (UCSD) campus in the early 1980s; since that time this population has remained isolated geographically and genetically, about 70 km from the nearest breeding population with low levels of immigration (Rasner et al. 2004
; Yeh and Price 2004
). Several morphological and behavioral changes have occurred in this short period of time, including cessation of migration, reduced wing length, reduction of a sexually selected plumage characteristic, shifts in reproductive behavior including a longer breeding season, reduced territorial aggression, and increased exploratory boldness compared with juncos in the presumptive ancestral range (Rasner et al. 2004
; Yeh 2004
; Yeh and Price 2004
; Newman et al. 2006
; Yeh et al. 2007
; Price et al. 2008
; Atwell JW, unpublished data). Data from common garden experiments suggest that changes in plumage traits and exploratory boldness may have a genetic basis (Yeh 2004
; Atwell JW, unpublished data).
Juncos in this study were originally captured from the UCSD campus (lat 32°40′N, long 117°10′W; elevation 30 m) and the Laguna Mountain Recreation Area in the Cleveland National Forest (lat 32°52′N, long 116°25′W; elevation 1700 m). These birds were captured as juveniles (recognizable by a distinct plumage) soon after they became independent (30–40 days posthatch) in June and July 2007, using the same capture methods (mist nets and walk-in traps) for both populations. After capture, juveniles were housed in flocks in temporary outdoor aviaries in suburban San Diego, CA until mid-July 2007, then shipped via air cargo to the Kent Farm Bird Observatory (KFBO) indoor aviaries at Indiana University in Bloomington, IN. At KFBO, the birds were segregated by population into 2 large identical windowless aviary rooms (6.4 m L × 3.2 m W × 2.4 m H) with equivalent densities (~1 bird/m2). Within each room, birds were housed in cages in mixed-sex pairs. Birds were segregated by population but otherwise held in identical conditions. The light schedule was set to match the photoperiodic schedule of the native breeding latitude of the 2 populations (which is the same). Temperature was maintained at 60–65 °F. Birds were given ad libitum access to water, seed, fruit, and mealworms in each cage. All aviary rooms had equivalent exposure to human researchers and animal care staff.
Five times during a 2-week period (at 2- to 3-day intervals) in June 2008, we repeatedly sampled preen oil from 26 captive juncos of both sexes from the 2 populations: from Laguna Mountain, 8 females and 6 males and from UCSD, 6 females and 6 males. Birds were sampled in a random order on each sampling day. In order to minimize handling stress during preen oil collection, we captured individuals from cages by darkening the lights in the room and quickly catching the target individual by hand and then keeping handling times to under 5 min. All birds were subjected to similar handling procedures. We collected preen oil by gently pressing a 100-μl glass micropipette tube (Drummond Scientific Company, Broomall, PA) against the uropygial gland and rubbing until a small amount (1–3 mg) of preen oil was secreted. Once collected, preen oil samples were stored at −20 °C until analyzed using gas chromatography–mass spectrometry (GC-MS) and gas chromatography–atomic emission detection (GC-AED).
Using a Teflon plunger, we pushed a thawed preen oil sample into a cleaned 20-ml glass vial and added 2.0 ml of water (high-purity OmniSolv, EMD Chemicals, Inc., Gibbstown, NJ), 100 mg of ammonium sulfate (99.99 + % from Sigma-Aldrich, St Louis, MO), and an internal standard (8 ng of 7-tridecanone, Sigma-Aldrich) dissolved in 5 μl of methanol (Baker Analyzed, Mallinckrodt Baker, Inc., Phillipsburg, NJ). Volatile compounds were extracted with the Twister stir bar (10 × 0.5 mm polydimethylsiloxane) for 60 min (Gerstel GmbH, Mülheim an der Ruhr, Germany). After extraction, the stir bar was rinsed with high-purity OmniSolv water, dried with a paper tissue, and placed in the thermal desorption autosampler tube.
Quantitative analysis was performed using the Agilent 6890N gas chromatograph connected to a 5973i MSD mass spectrometer (Agilent Technologies, Inc., Wilmington, DE) with the thermal desorption autosampler and cooled injection system (TDSA-CIS 4 from Gerstel). Positive electron ionization mode at 70 eV was used with a scanning rate of 2.47 scans/s over the mass range of 41–350 amu. The mass spectrometric detector (MSD) transfer line temperature was set at 280 °C. The ion source and quadrupole temperatures were set at 230 °C and 150 °C, respectively. The separation capillary was DB-5MS (20 m × 0.25 mm, inner diameter [i.d.], 0.25-μm film thickness) from Agilent (J&W Scientific, Folsom, CA). Samples were thermally desorbed in a TDSA automated system, followed by injection into the column with a cooled injection assembly, CIS-4. TDSA operated in a splitless mode and the temperature program for desorption was 20 °C (0.5 min), then 60 °C/min to 250 °C (3 min). Temperature of the transfer line was set at 280 °C. CIS was cooled with liquid nitrogen to −80 °C. After desorption and cryotrapping, CIS was heated at 12 °C/s to 270° C, with a hold time of 12 min. CIS inlet was operated in the solvent vent mode, a vent pressure of 9.1 psi, a vent flow of 50 ml/min, and a purge flow of 50 ml/min. The temperature program in the GC operation was 50 °C for 2 min, then increasing to 200 °C at the rate of 3 °C/min (hold time: 12 min). The carrier gas head pressure was 9.1 psi (flow rate, 1.1 ml/min at the constant flow mode).
GC–atomic emission spectrometry
Qualitative element-selective compound profiling was performed using a GC 6890 instrument equipped with an atomic emission detection system (AED, model G2350A) from Agilent Technologies and a thermal desorption autosampler-cooled injection system (TDSA-CIS-4 from Gerstel). The separation capillary was HP-5MS (30 m × 0.25 mm, i.d., 0.25-μm film thickness) from Agilent. Samples were thermally desorbed in a TDSA automated system, followed by injection into the column with a cooled injection assembly under the same conditions as described above for the GC-MS analysis, except that the CIS was cooled with liquid nitrogen to −60 °C. Temperature of the transfer line was 280 °C. The emission lines for carbon (193 nm), sulfur (181 nm), and nitrogen (174 nm) were monitored during the atomic plasma emission detection.
Among approximately 100 compounds detected in the preen oil, about 40 components of chromatographic profiles were tentatively identified. All major compounds were positively identified by comparison with standard substances of known mass spectra and retention times. Peak areas of the identified compounds were used for quantitative comparisons among the groups. Peak areas were integrated either from the total ion current (TIC) profiles or from the postrun single ion current (SIC) profiles at m/z 55, m/z 58, and m/z 60. Peak areas of the internal standard were integrated from the postrun m/z 113 profiles. Peak areas of the compounds of interest were normalized by dividing each peak area by that of the internal standard in corresponding runs. Relative standard deviation (a measure of reproducibility) of the internal standard peak area was 13% (n = 12).
The GC-AED system provides about 100 times more sensitivity for sulfur-containing organic compounds at the sulfur emission line (181 nm) than GC-MS measurements. Despite this ultrahigh detection sensitivity, no consistently appearing sulfur-containing compounds were detected in the junco preen oil samples.
We focused on 19 volatile compounds, quantified by GC-MS, that were previously found to vary seasonally in juncos and to increase during the breeding season (Soini et al. 2007
; Soini HA and Whittaker DJ et al., unpublished data), suggesting their possible role in reproductive behavior. We examined individual “volatile profiles” of compounds by testing for repeatability and individual differences in the relative proportions of each compound (Svensson et al. 1997
; Miklas et al. 2000
). GC-MS peak areas were measured, and for each volatile compound, the observed GC-MS peak area was converted to a percentage of the total observed peaks. Because the proportion data are not normally distributed, we then logit transformed the data by taking the natural logarithm of (p
/(1 − p
)), where p is the proportion (Armitage and Berry 1994
We calculated repeatability (r
) of the relative proportions of individual volatile compounds in repeated preen oil samples from the same individual using the following formula:
is the among-groups variance component (variation among individuals), MSW
is the within-groups variance component (variation within individuals), and n0
is the sample size (Lessells and Boag 1987
After determining that relative measurements of volatile compounds were highly repeatable for each individual, we averaged the 5 measurements to obtain a single measurement for each volatile compound for each individual to avoid pseudoreplication before proceeding. We conducted a principal components analysis (PCA) (SPSS 16.0) using 17 of the identified volatile compounds (2 compounds, nonanoic acid, and decanoic acid, were below detectable levels in several individuals and were excluded from the analysis) and rotated the component matrix to maximize variance (varimax rotation). We then tested for differences between groups by conducting a multivariate analysis of variance (MANOVA) using sex and population as fixed factors and the synthetic variables generated from the PCA as the dependent variables.