Ecological Applications, 19(5), 2009, pp. 1264?1273  2009 by the Ecological Society of America Mesoscale patterns of altitudinal tenancy in migratory wood warblers inferred from stable carbon isotopes GARY R. GRAVES1,3 AND CHRISTOPHER S. ROMANEK2 1Department of Vertebrate Zoology, MRC-116, National Museum of Natural History, Smithsonian Institution, P.O. Box 37012, Washington, D.C. 20013-7012 USA 2Department of Geology and Savannah River Ecology Laboratory, Drawer E, Aiken, South Carolina 29802, USA, and University of Georgia, Athens, Georgia 30602 USA Abstract. We analyzed carbon isotope ratios (d13C) of liver and pectoral muscle of Black- throated Blue Warblers (Dendroica caerulescens) to provide a mesoscale perspective on altitudinal tenancy in the Appalachian Mountains, North Carolina, USA. Movements of males are poorly understood, particularly the degree to which yearlings (?rst breeding season) and older males (second or later breeding season) wander altitudinally during the breeding season. Liver and muscle d13C values of warblers exhibited signi?cant year and altitude effects, but yearling and older males were isotopically indistinguishable. Liver d13C values increased with altitude at the rate of ;0.5? per 1000 m. The altitudinal lapse rate of muscle d13C (;1.1? per 1000 m) was nearly identical to the average rate of increase reported in several groups of C3 plants (;1.1? per 1000 m). This suggests that the majority of males foraged within relatively narrow altitudinal zones during the breeding season. We caution, however, that the discrimination of altitudinal trends in carbon isotope ratios depends on relatively large multiyear samples. Given the scatter in data, it is unlikely that individuals can be accurately assigned to a particular altitude from carbon isotope values. Rapid adjustment of liver and muscle d13C values to local altitudinal environments is consistent with the results of experimental dietary studies that show carbon turnover rates are relatively rapid in small migratory passerines. In a broader context, carbon isotope data have been increasingly used as proxies for wintering habitat use of Nearctic?Neotropical migratory passerines. However, tissues with high metabolic rates are unlikely to retain much isotopic signal of wintering habitat use by the time migrants reach their breeding territories. Key words: altitudinal tenancy; Appalachian Mountains, North Carolina, USA; Black-throated Blue Warbler; Dendroica caerulescens; isotope turnover rates; migratory birds; stable carbon isotopes. INTRODUCTION The capacity to track the movements of migratory birds in real time throughout their annual cycle is arguably the Holy Grail of avian ecology. Although satellite telemetry and archival tags promise to revolu- tionize knowledge of dispersal and migratory behavior of larger avian species such as albatross (Jouventin and Weimerskirch 1990) and geese (Gudmundsson et al. 1995), the vast majority of migratory songbirds are too small to carry powerful transmitters or data recorders. Banding programs have been the principal method for studying dispersal and migration of songbirds for the past century (B?nl?kke et al. 2006). The substantial up- front investment of time and ?nancial resources required to mark individuals is a major drawback of mark? recapture protocols, but the principal weakness is that population-level patterns of dispersal and connectivity between breeding and wintering areas are rarely discernable because recovery rates of songbirds are vanishingly low away from initial capture sites. Thus there is an urgent need to develop new methods of tracking population movements of migratory passerines. Stable isotope analysis has shown some promise as a tool for elucidating dispersal patterns (Graves et al. 2002, Hobson et al. 2003, Hobson et al. 2004, Hobson 2005b) and the geographic origins of avian populations from ?eld sampling of unmarked individuals (Cham- berlain et al. 1997, Hobson and Wassenaar 1997, Hobson 1999, Bearhop et al. 2005). The power of stable isotope analysis in ecological research lies in the predictable relationship between assimilated food and water resources and the isotopic composition of biological tissues (DeNiro and Epstein 1978, Estep and Dabrowski 1980, DeNiro and Epstein 1981, Macko et al. 1983, Hobson et al. 1999). In migration studies, the assignment of individuals to a particular geographic locality depends on the robust mapping of isotopic gradients (Poage and Chamberlain 2001, Meehan et al. 2004, Bowen et al. 2005) as well as the spatial distribution of isotopically distinctive food resources within the potential geographic range of the species Manuscript received 20 May 2008; revised 23 September 2008; accepted 6 October 2008. Corresponding Editor (ad hoc): S. R. Beissinger. 3 E-mail: gravesg@si.edu 1264 (Romanek et al. 2000, Wassenaar and Hobson 2000, Chamberlain et al. 2005, Cerling et al. 2006). Despite recent methodological advances in isotope ecology, continental-scale analyses of migratory birds continue to yield no better than coarse isotopic discrimination of populations, even when combinations of elements are examined (Rubenstein et al. 2002, Royle and Rubenstein 2004, Bowen et al. 2005, Hobson 2005a, Passey et al. 2005, Wunder et al. 2005, Rocque et al. 2006, Kelly et al. 2008, Wunder and Norris 2008). Failure to achieve ?ner spatial resolution may stem, in part, from the confound- ing effects of topography on continental isotope gradients of hydrogen, oxygen, and carbon, the most commonly investigated elements in avian migration studies (Hobson 2005a, West et al. 2006). Precipitation and surface water exhibit progressively lower dD (ratio of deuterium to protium) and d18O values with increasing altitude and latitude and distance from coastlines (Dansgaard 1964, Siegenthaler and Oeschger 1980, Poage and Chamberlain 2001, Meehan et al. 2004, Bowen et al. 2005). d13C values increase with altitude and latitude in C3 plants as a consequence of pressure and temperature effects on carboxylation ef?ciency and other factors during photosynthesis (Ko?rner et al. 1988, Vitousek et al. 1990, Ko?rner et al. 1991, Marshall and Zhang 1994, Sparks and Ehleringer 1997, Hultine and Marshall 2000). While more detailed isotope base maps for topographically complex regions will improve the speci?city of isotopic interpretations, one impediment to achieving ?ner isotopic resolution of breeding bird populations is the dif?culty of obtaining suf?cient numbers of specimens strati?ed by sex, age class, molt, and geographic location. Intensive multiyear investiga- tions conducted in relatively small catchments suggest that ?ner isotopic resolution at mesoscales may be possible if altitudinal effects and other within-popula- tion sources of isotopic variation are identi?ed (Graves et al. 2002). The Black-throated Blue Warbler (Dendroica caer- ulescens) breeds in cool deciduous and mixed deciduous? coniferous forests in eastern North America and winters in the Caribbean basin, primarily in the Greater Antilles (Holmes 1994, Rubenstein et al. 2002). Populations breeding south of 408 N latitude are restricted to higher altitudes in the Appalachian Mountains. Graves et al. (2002) examined altitudinal variation of carbon isotope ratios (13C/12C) in feathers of males breeding in the Big Santeetlah Creek watershed, a relatively small but topographically complex catchment in western North Carolina, USA. The Big Santeetlah Creek population is part of a larger metapopulation in the southern Appalachian Mountains (105?106 individuals; G. R. Graves, unpublished data), where it is one of the most common breeding species between 800 m and 1450 m above sea level (a.s.l.) (Wilcove 1988, Graves 1997b, Haney et al. 2001). In the southern Appalachians, males return to breeding territories (0.75?3.0 ha) at lower altitudes (,800 m a.s.l.) as early as 1 May, but settlement at higher altitudes (.1300 m a.s.l.) may be delayed by two to three weeks. The staging areas for individuals that eventually occupy territories at higher altitudes are unknown. The diet of nestlings and adults during the breeding season is predominately lepidopter- an larvae gleaned from the foliage and twigs of trees and shrubs (Rodenhouse and Holmes 1992, Holmes 1994). This small migrant (9?10 g) undergoes a complete molt in July and August before fall migration to the Caribbean basin, such that ?ank feathers sampled during the current breeding season were grown on or near their breeding or natal territories during the previous year (Holmes 1994, Graves 1997a). In theory, carbon isotope pro?les of feather keratin re?ect the diet ingested during June and July and provide a tool for tracking the year-to-year movements of individuals along altitudinal gradients in C3 forests. Carbon isotope ratios for yearling males (?rst breeding season) and older males (second or later breeding season) exhibited divergent altitudinal patterns. Whereas feather d13C values from yearling males were uncorrelated with altitude, most likely re?ecting natal dispersal (i.e., movement from hatching site to ?rst breeding territory), feather d13C values for older males increased with altitude at the rate of ;1.3? per 1000 m, which approximates the lapse rate (;1.1? per 1000 m) observed in several groups of C3 plants along altitudinal gradients (Ko?rner et al. 1991). This suggests that older males exhibit a signi?cant degree of breeding season philopatry to narrow altitudinal zones, if not to individual territories. However, the range of feather d13C values reported from the watershed nearly brack- eted the range of feather d13C values observed in breeding populations sampled from Georgia to New Brunswick (118 of latitude). These ?ndings suggest that the isotopic detection of long-distance dispersal, a process facilitated by the identi?cation of statistical outliers or higher isotopic variance in local populations (Hobson et al. 2004, M?ller and Hobson 2004, M?ller et al. 2006) may be complicated by local isotopic variation in topographically complex regions. On the other hand, the lapse rate of d13C values observed along altitudinal gradients in C3 plants and feather keratins suggests that under certain circumstances the carbon isotope pro?les of metabolically active tissues may provide useful information on altitudinal tenancy patterns in local populations. Knowledge of tenancy patterns of Black-throated Blue Warblers during the peak of the breeding season has been limited to observations of marked individuals on small, comparatively level, study plots in the Hubbard Brook Experimental Forest, New Hampshire, USA (Holmes et al. 1992, Marra and Holmes 1997, Webster et al. 2001, Sillett et al. 2004). This species, however, reaches its greatest abundance in the southern Appalachian Mountains where the altitudinal amplitude of local breeding populations may exceed 800 m (Graves 1997b, Graves et al. 2002). Male behavior in the July 2009 1265STABLE CARBON ISOTOPES IN WOOD WARBLERS topographically rugged Appalachians is poorly under- stood; particularly the degree to which yearlings (?rst breeding season) and older males (second or later breeding season) wander altitudinally during the nesting season after the establishment of territories. Differences in the breeding biology of yearling and older males suggest that patterns of altitudinal tenancy may be age related. For instance, yearlings arrive later on the breeding grounds than older males (Hubbard 1965); they are behaviorally subordinate (Holmes et al. 1996) and less likely to occupy high-quality territories than older males (Holmes et al. 1996). Once settled, both yearling and older males defend small territories and are thought to be relatively sedentary, except for a small subset of unmated males (???oaters??) that search for females and undefended territories (Holmes et al. 1992, Marra and Holmes 1997). In most passerine species, ?oaters are more likely to be yearling males (Smith and Arcese 1989), which implies that the pattern of altitudinal tenancy in yearling warblers may differ fundamentally from that of older males. In this study, we present the results of an eight-year investigation of d13C values in muscle and liver samples of the Black-throated Blue Warbler, a study designed to examine mesoscale patterns of altitudinal tenancy during the peak of the breeding season (June) in the southern Appalachian Mountains. Because animal tissues exhibit a range of isotopic turnover rates, depending on the metabolic rate of the tissue and the element of interest, analyses of two or more tissue types from an individual may provide information on foods assimilated over different time scales. Analyses of liver and pectoral muscle d13C values provide differential pro?les of isotope assimilation from warbler diets over comparatively short time frames (days to weeks). We hypothesized that liver and muscle of yearlings and older males would display similar isotopic patterns of variation if altitudinal movements (i.e., patterns of altitudinal tenancy) of the two age classes were similar. By sampling territorial males over eight breeding seasons from a relatively small catchment, we were able to examine age- speci?c and year effects while controlling for the in?uence of altitude on carbon isotope signatures. METHODS Study site This study was conducted in the Big Santeetlah Creek watershed (358210 N, 848000 W) on the eastern slope of the Unicoi Mountains, the highest subsidiary mountain range in the Appalachians south of the Little Tennessee River (Graves et al. 2002). This forested watershed (680? 1689 m a.s.l., 5350 ha) in Graham County, North Carolina, is embedded in the largest contiguous tract of montane forest in eastern North America and supports one of the most taxonomically diverse woody ?oras north of Mexico (Lorimer 1980). The terrain is steep, and slopes commonly vary from 20% to 40%. The greatest dimensions of the study area are ;11.3 km (east?west axis)3;8.0 km (north?south axis). Forestry practices since the 1920s have resulted in a mosaic of relict old-growth trees and even-aged stands (x?? 70 6 41 years [x??mean age of forest stands 6 SD]; n? 100 stands) of hardwood?hemlock forest dominated by C3 plants. Cove and streamside forest in the Big Santeetlah watershed is dominated by hemlock (Tsuga canadensis), tulip poplar (Liriodendron tulipifera), sugar maple (Acer saccharum), red maple (Acer rubrum), beech (Fagus grandifolia), northern red oak (Quercus rubra), yellow birch (Betula alleghaniensis), sweet birch (Betula lenta), silverbell (Halesia carolina), and white basswood (Tilia heterophylla). Concentrations of chestnut oak (Quercus prinus) are found on drier slopes and ridges. American chestnut (Castanea dentata) was a major component in this community prior to the chestnut blight (Braun 1950, Lorimer 1980). Witch hazel (Ham- amelis virginiana), striped maple (Acer pensylvanicum), mountain maple (Acer spicatum), ?owering dogwood (Cornus ?oridana), and alternate-leaf dogwood (Cornus alternifolia) are important subcanopy species. The understory is dominated by ??evergreen?? thickets of rosebay (Rhododendron maximum) and mountain laurel (Kalmia latifolia). Important components of the shrub layer that provide nesting sites for Black-throated Blue Warbler include hydrangea (Hydrangea arborescens), mapleleaf viburnum (Viburnum acerifolium), hobble bush (Viburnum alnifolium), smooth allspice (Calycan- thus fertilis), and ?owering raspberry (Rubus oderatus), as well as tree saplings. Grassy balds occur at the summit of peaks (.1600 m), but C4 crops have not been cultivated in the watershed in .70 years. Population sampling Males (n? 348) were collected under state and federal licenses for multiple research purposes (Graves 1997b, Graves et al. 2002, Rubenstein et al. 2002, Graves 2004, Fallon et al. 2006, Grus et al. 2009) during eight consecutive breeding seasons (1995?2002) along tran- sects spanning the altitudinal range (750?1545 m) of the TABLE 1. Summary statistics for Black-throated Blue Warblers (Dendroica caerulescens) collected during eight consecutive breeding seasons in the Big Santeetlah Creek watershed, western North Carolina, USA. Year Collection date Altitude, mean 6 SD (m) No. yearling males No. older males 1995 18?23 June 1119 6 168 4 35 1996 12?20 June 1167 6 185 11 36 1997 11?19 June 1152 6 191 9 34 1998 10?18 June 1152 6 187 12 23 1999 10?20 June 1115 6 207 19 24 2000 10?18 June 1128 6 185 20 25 2001 9?17 June 1148 6 198 26 24 2002 11?18 June 1160 6 185 22 24 Note: Mean altitude is expressed as meters above sea level. GARY R. GRAVES AND CHRISTOPHER S. ROMANEK1266 Ecological Applications Vol. 19, No. 5 species in the Big Santeetlah Creek watershed (Table 1). The altitude of all collecting sites was determined with a Thommen altimeter (Revue Thommen, Waldenburg, Switzerland) calibrated from landmarks on U.S. Geo- logical Survey 7.5-minute topographic maps. Year-to- year variation in the altitudinal distribution of collected specimens was statistically insigni?cant (ANOVA, F7, 340 ? 0.46, P? 0.87; Table 1). Males collected in this study responded to playback of recorded songs and exhibited territorial behavior. Although females were observed on many of the territories, we did not systematically attempt to locate females or nests. The sampling period (9?23 June) coincided with the overlap of the ?edging period for the ?rst brood and nest-building and egg- laying for the second brood. Older males were prefer- entially collected from 1995 through 1998, whereas a more even balance of yearlings and older males was obtained in the latter years of the study. Annual standardized censuses along a 14.5-km altitudinal transect indicated that the removal of males in the watershed had no demonstrable effect on the census population. Specimens were packaged whole in multiple layers of insulating tissue paper and aluminum foil and frozen within 30 min of death in liquid nitrogen. Voucher specimens (rounded skins, partial skeletons, tissues, stomach contents) were deposited in the research collection of the National Museum of Natural History, Smithsonian Institution. Two age classes of males were distinguished by plumage characters (Graves 1997a): (1) yearlings hatched the previous year (?rst alternate plumage, or SY in banding terminology), and (2) older individuals in their second or later breeding season (de?nitive alternate plumage, or ASY in banding terminology) (Pyle 1997). Yearling males were identi?ed by the olive-green (rather than blue) edges of their alulae and primary coverts and a suite of other characters (Graves 1997a). Isotopic turnover rates Feeding trials and isotope assays of tissues have been conducted on several large-bodied avian species (Hob- son and Clark 1992, 1993, Haramis et al. 2001, Bearhop et al. 2002). However, only three studies of isotope turnover rates have been conducted on small migratory passerines (,20 g) with higher metabolic rates (Hobson and Bairlein 2003, Pearson et al. 2003, Podlesak et al. 2005), and these studies were limited to examinations of carbon and nitrogen isotopes of whole blood or blood components and feathers. Experimental dietary studies with captive quail (Coturnix japonica, ;115 g) conclud- ed that the half-lives of carbon isotopes were ;2.7 d for liver, ;11.4 d for whole blood, and ;12.4 d for pectoral muscle (Hobson and Clark 1992). Whole blood of smaller, captive Yellow-rumped Warblers (Dendroica coronata, 11 g; Pearson et al. 2003) and Garden Warbler (Sylvia borin, 20 g; Hobson and Bairlein 2003) had carbon half-lives of 3.9?6.1 and 5.0?5.7 d, respectively. These studies suggest that carbon isotopes in the whole blood of wood warblers may exhibit nearly complete turnover (.98%) in as little as 24 days (;6 half-lives). Carbon isotope turnover rates in liver and pectoral muscle of small migratory passerines are unknown. However, given the observation that turnover rates for whole blood and pectoral muscle in larger birds are roughly equivalent and turnover rates for liver are about four times faster than for muscle (Hobson and Clark 1992), we estimated a turnover rate of ;1.0?1.5 d for liver and ;4?6 d for pectoral muscle of small migratory passerines. As such, the carbon isotope signatures of liver and pectoral muscle of the warblers sampled in this study undoubtedly re?ect the diet assimilated on breeding territories rather than during migration or on the wintering grounds. Isotope analyses Liver and pectoral muscle samples were freeze-dried, soaked in a 2:1 chloroform :methanol mixture for 24 h to remove lipids (Hobson and Welch 1992), rinsed with methanol, and then oven-dried at ;458C. Prior to isotope analyses, samples were homogenized by grinding with a mortar and pestle. After grinding, 2?3 mg of each sample were weighed to the nearest 61 lg in a precleaned tin capsule. Capsules were then sealed and placed in the autosampler of a Carlo Erba Elemental Analyzer (NA 2500; Thermo Electron, Milan, Italy) attached to a continuous-?ow isotope ratio mass spectrometer (Finnigan DeltaPLUS XL, Finnigan-MAT, San Jose, California, USA) for carbon and nitrogen isotope analysis. Samples were converted to CO2 and N2 in oxidation?reduction furnaces, separated by gas chromatography, and then measured for 13C/12C and 15N/14N ratios on the mass spectrometer. An internal N2(g) working standard was admitted prior to the introduction of each sample and a CO2(g) standard was admitted at the conclusion of each combustion for calibration to AIR (nitrogen) and Vienna PeeDee Belemnite (V-PDB) (carbon) interna- tional standards (Mariotti et al. 1980, Coplen 1996). Stable nitrogen isotope pro?les of warbler tissues were uncorrelated with altitude (Appendix A) and will not be discussed further. Stable isotope ratios are reported in per-mil units (?) using standard delta (d) notation (Craig 1961). External working standards of dog?sh muscle and liver (DORM- 2) were reproducible to ,0.2? (SD) per run for d13C. All isotope analyses were performed at the University of Georgia?s Savannah River Ecology Laboratory. The d13C values were tested for goodness of ?t to a normal distribution with the Lilliefors test. We used analysis of covariance (ANCOVA) to investigate the effects of categorical (age class, year) and continuous variables (altitude, day of year) on isotope values for pectoral muscle and liver (ANOVA module of SYSTAT Version 11). We used paired t tests to compare mean isotope values of liver and pectoral muscle. We used ordinary least squares regression (OLS) to investigate the July 2009 1267STABLE CARBON ISOTOPES IN WOOD WARBLERS relationship between isotope values and selected inde- pendent variables. All P values are two tailed (r? 0.05). RESULTS Stable carbon isotopes of liver The liver d13C values from the pooled sample of yearling and older males (n? 8 yr) ranged from25.5 to 19.6 (x ? 23.0? 6 0.8? [mean 6 SD]; n ? 330). Year-to-year differences (e.g., 1997 to 1998) in mean liver d13C values ranged from 0.1? to 0.9? (Fig. 1). Mean liver d13C values were signi?cantly higher than muscle d13C values for both yearling and older males (Table 2, Fig. 2). Liver and muscle d13C values were positively correlated in yearlings (OLS: R2 ? 0.27, P , 0.0001; n?114) and in older males (OLS: R2? 0.30, P, 0.0001; n ? 212). The relationship between liver d13C values and altitude showed considerable year-to-year variation, and signi?cant correlations (OLS) were observed in only three of eight years (Fig. 3, Appendix B). In the pooled sample (n ? 8 yr), liver d13C values increased with altitude at the rate of ;0.5? per 1000 m (liver d13C ? 23.54 ? 0.0005 [altitude, m]). In ANCOVA, liver d13C values exhibited signi?cant altitude (P ? 0.005) and year effects (P , 0.0001) but no age class or seasonal (day of year) effects (Table 3). Liver d13C values were uncorrelated with feather d13C values reported in Graves et al. (2002) for feathers grown during the previous molt in yearling (OLS: R2 , 0.01, P ? 0.69; n ? 115) and older males (OLS: R2 , 0.01, P ? 0.23; n ? 215). Stable carbon isotopes of pectoral muscle The muscle d13C values for the pooled sample of yearlings and older males ranged from 25.7? to 19.8? (x ?23.2? 6 0.7? [mean 6 SD]; n ? 331). Year-to-year differences in mean muscle d13C values ranged from 0.1? to 0.5? (Fig. 1). The relationship between muscle d13C values and altitude exhibited substantial year-to-year variation (Fig. 3, Appendix C). Slope coef?cients (OLS) were positive during all breeding seasons, but signi?cant correlations were observed in only ?ve of eight years. In the pooled sample (n ? 8 yr) of yearling and older males, muscle d13C values increased at a rate of ;1.1? per 1000 m (muscle d13C?24.46?0.0011 [altitude, m]). ANCOVA indicated that muscle d13C values were signi?cantly in?uenced by altitude (P , 0.0001) and year effects (P , 0.0001) but not by age class or seasonal effects (Table 3). Muscle d13C values were uncorrelated with feather d13C values reported in Graves et al. (2002) for feathers grown during the previous year in yearlings (OLS: R2 , 0.01, P ? 0.53; n ? 116) and in older males (OLS: R2 ? 0.02, P , 0.06; n ? 215). DISCUSSION Altitude effects in isotope signatures Signi?cant altitude effects for liver and muscle d13C values were consistent with the relationship previously described for feather d13C from older males in the Big Santeetlah Creek watershed (Graves et al. 2002). In the pooled sample of yearling and older males, liver d13C values increased with altitude at the rate of ;0.5? per 1000 m, whereas muscle d13C values increased at a rate of ;1.1? per 1000 m. The difference in the altitudinal lapse rates of muscle and liver d13C values may be related to the disparity in isotopic turnover rates of liver and muscle. The lapse rate of muscle d13C values with FIG. 1. Box plots depicting the annual ?uctuation of d13C values for liver and pectoral muscle in yearling males (right- hand box for each year) and older males (left-hand box) of the Black-throated Blue Warbler (Dendroica caerulescens) from the Big Santeetlah Creek watershed, western North Carolina, USA. Horizontal lines within boxes represent the median, and whiskers represent the range of data values. A few outliers were omitted from the ?gure. TABLE 2. Summary statistics for d13C values of liver and pectoral muscle (mean6 SD) of male Black-throated Blue Warblers collected in the Big Santeetlah Creek watershed. Age class Liver Pectoral muscle t P Yearlings 23.1? 6 0.9? (115) 23.3? 6 0.7? (116) 2.34 0.02 Older males 22.8? 6 0.8? (215) 23.2? 6 0.8? (215) 7.26 ,0.0001 Note: Parenthetical values represent n, the number of individual birds.  Paired t test. GARY R. GRAVES AND CHRISTOPHER S. ROMANEK1268 Ecological Applications Vol. 19, No. 5 -26 0)0)0)0)0)000 ) ) ) ) ) T-I-I-T-T-CMCMCM altitude was comparable to that observed in feather d13C values in older males (;1.3? per 1000 m) and nearly identical to the average rate of increase reported in several groups of C3 plants (;1.1? per 1000 m) along altitudinal transects (Ko?rner et al. 1991). The signi?- cantly lower F ratios observed for altitudinal effects in liver d13C (F ? 7.92) compared to muscle d13C (F ? 33.33) suggest that liver data may more closely track ?uctuating isotope signals in lepidopteran prey at any given altitude. Temporal variability in the isotopic composition of diet is also suggested by the surprisingly low correlation between liver and muscle d13C values in yearlings (R2? 0.27) and older males (R2? 0.30) and the year-to-year ?uctuations in correlation coef?cients (Appendices B, C). Regardless of the nuances of the carbon isotope record, the rapid adjustment of d13C values to local altitudinal environments is consistent with the hypoth- esis that carbon turnover rates are relatively rapid in liver and pectoral muscle of small migratory passerines. Liver and muscle d13C values (measured in year t ? 1) were uncorrelated with feather d13C values (feathers grown in year t) in yearlings and older males. This ?nding is unsurprising because yearling wood warblers rarely breed near their natal territories (Nolan 1978, Holmes 1994) and because annual mortality rates of adults may exceed 40% (Nolan 1978, Holmes 1994, Holmes et al. 1996). Consequently, as few as half the territories in any given breeding season are occupied by incumbents. Altitudinal tenancy during the breeding season Similar altitudinal trends of d13C variation in liver and pectoral muscle of yearlings and older males suggest that the majority of territory holders of both age classes are relatively sedentary during the peak of the breeding season. In other words, yearlings appeared to be no more likely than older males to wander altitudinally in the days and weeks leading up to the collection date. However, there was considerable scatter in the data and some curious outliers among yearlings and older males (see Fig. 3). We suspect the majority of scatter in d13C data is related to annual variation in climate (expressed as year effects) and the topographic complexity of territories. Owing to the steep terrain in the Santeetlah Creek watershed, the altitudinal range of male territories typically spans 10?20 m and may exceed 50 m on some territories. A multitude of factors that affect d13C values in C3 plants (and isotope signatures of lepidopteran larvae), such as moisture availability and irradiance, vary with slope and aspect (Ehleringer et al. 1986, Zimmerman and Ehleringer 1990, Hanba et al. 1997, Saurer et al. 1997). We consider several other potential sources of data scatter as less likely. For example, some outliers may represent territorial males that were drawn signi?cantly upslope or downslope beyond the bound- aries of their territories by the broadcast of taped songs. Alternatively, these data points may represent wander- ing ?oaters that responded to playback of songs and brie?y exhibited territorial behavior at the collection site. A fourth possibility is that outliers represent territorial males that were intercepted as they brie?y explored outlying territories, perhaps searching for extra-pair copulations (Webster et al. 2001). Such reconnaissance ?ights traversing 100?200 m of altitude would require only a few minutes to complete and would be very dif?cult to detect through direct observation, standard mark?recapture methods, or radio telemetry. Despite the many sources of isotopic noise in the data, the signi?cant correlation between d13C values of metabolically active tissues and altitude suggest that carbon isotopes may have some use in evaluating aggregate patterns of tenancy along altitudinal gradi- ents. We caution, however, that the altitudinal signal of stable carbon isotopes in warbler tissues is relatively weak and that the discernment of mesoscale patterns depends on relatively large sample sizes obtained over multiple breeding seasons. Our data indicate that analyses limited to a single breeding season can yield results that are at odds with isotopic patterns that emerge over time periods equivalent to the maximum life span of passerine birds. Given the scatter in data, it is unlikely that individuals can be accurately assigned to a FIG. 2. Distribution of differences in carbon isotope values (d13C for liver  d13C for muscle) within individual Black- throated Blue Warblers of two age groups. July 2009 1269STABLE CARBON ISOTOPES IN WOOD WARBLERS 0) .a E =3 (D .a E Older males 60 _ 40 - H 20 - i?, rt-Hl _L JTln. 80 60 40 20 0 Yearlings I r-fTTTT--L--iIl -4 -3 -2 -1 0 1 2 3 gi=C liver - 8'*C muscle (%o) particular altitude solely from carbon isotope values. Future studies should explore the feasibility of combin- ing carbon, oxygen, and hydrogen isotope data from a variety of tissues in altitudinal assignment models. Implications for connectivity analysis of wintering and breeding populations of migratory birds Stable isotope data are frequently used as proxies for wintering habitat quality of birds sampled on breeding territories or captured during migration (Marra et al. 1998, Norris et al. 2003, Bearhop et al. 2004). The validity of such studies depends on the isotope turnover rates of assayed tissues and the duration of migration. Feeding experiments indicate that isotopic turnover rates in whole blood and plasma of small migratory birds (,20 g) are rapid, perhaps too rapid to provide much isotopic signal of wintering habitat for species sampled on breeding territories at high latitudes (Hobson and Bairlein 2003, Pearson et al. 2003, Hob- son 2005a, Mazerolle and Hobson 2005). Nearctic? Neotropical migratory passerines, such as the Black- throated Blue Warbler, typically take several weeks to reach their breeding grounds (Cooke 1888, 1915). For example, median capture dates for 15 species of FIG. 3. Relationship between (A) d13C muscle and (B) d13C liver values, paired for each year, with altitude of breeding territories of Black-throated Blue Warblers. Yearlings and older males are represented respectively by 3?s and open circles. See Appendix B (liver d13C) and Appendix C (muscle d13C) for slope coef?cients and intercepts of least squares regression lines. Altitude is expressed as meters above sea level. GARY R. GRAVES AND CHRISTOPHER S. ROMANEK1270 Ecological Applications Vol. 19, No. 5 -20 -22 1995 p -24 -26 A ?aa o^___J i i i i O ? 8 x Yearling males o Older males _l I I L_ -20 -22 -24 -26 1999 A i i i J^*( o 8 ^^t o i o i i B i i i si 9^-*-""" 5_3 ^ %%"W ?-^T ^? 0 i i i -20 1996 -22 O -24 -26 -20 -22 A ? go - - %W%P&8 "J5 1997 O -24 -26 -20 A 0 r< ? ?o CLS6& -8? r"&^ ^ O^ 0 X 1998 -22 o -24 -26 1 1 1 9-1 A o ___-c@K%ox ? o B ^8x4 }?L> 0 % 00 ^08 -20 -22 -24 -26 -20 -22 -24 -26 -20 -22 - 2000 1 1 A 1 1 ? *e ? ? ?o JOX; 0, Ox X % X ?p 0 0 X , B >W? o?xO n ^ X X .X)K % 0 0 (% 2001 A 1 1 1 . % . yg"^8@r^ 0 r\^? x. X B 1 - A^ - ^-S"; X *x 2002 ?o^^^ 6 8 10 12 14 16 6 8 10 12 14 16 Altitude (hundreds of meters) _ xo Oy * 00^ X 10 12 14 16 6 8 10 12 14 16 Altitude (hundreds of meters) migratory passerines at a banding station on the Gulf Coast of Louisiana were 22 days earlier than those recorded for the same species at two stations located approximately 2500 km to the north in Pennsylvania and Ontario (Marra et al. 2005). Nocturnally migrating passerines characteristically refuel at diurnal stopover locations, which could result in a stepwise change in the isotopic composition of metabolically active tissues during the transit (Hobson 2005a). The lengthy spring migration of Neartic?Neotropical migrant passerines suggests that the bulk of the carbon isotope signal in blood, liver, and muscle of individuals at the time of their arrival on their breeding territories re?ects food consumed en route. This implies that tissues with high metabolic rates sampled from migratory species on breeding territories have limited value as isotopic proxies of wintering habitat use. Tissues that lock in isotopic signals over a limited time frame such as toenail keratins and feathers, for species that molt on the wintering grounds, are much more likely to re?ect the isotopic composition of wintering-ground diet and habitats (Chamberlain et al. 2000, Bearhop et al. 2004, Mazerolle and Hobson 2005). ACKNOWLEDGMENTS We thank Lindy Paddock and Kenneth Cole for laboratory assistance. Seth Newsome, Mike Wunder, and an anonymous reviewer provided cogent reviews. Brian Schmidt, Chris Milensky, Phil Angle, Jim Dean, and Carla Dove prepared specimens, and Joe Bonnette, John Gerwin, Chuck Hunter, Glen McConnell, and Mark Robison helped with permits. Gary R. Graves was supported by the Alexander Wetmore Fund and the Biodiversity Surveys and Inventory Program of the National Museum of Natural History, Smithsonian Institution. Christopher S. Romanek was supported by the Department of Energy under Award Number DE-FC09-07SR22506 to the University of Georgia Research Foundation. Specimens were collected under the auspices of the Animal Care and Use Committee, National Museum of Natural History. Research permits were issued by the U.S. Fish and Wildlife Service, U.S. Forest Service (Department of Agriculture), and North Carolina Wildlife Resources Commission. LITERATURE CITED Bearhop, S.,W. Fiedler, R.W.Furness, S. C. Votier, S.Waldron, J.Newton,G. J. Bowen,P.Berthold, andK.Farnsworth. 2005. Assortative mating as a mechanism for rapid evolution of a migratory divide. Science 310:502?504. Bearhop, S., G. M. Hilton, S. C. Votier, and S. Waldron. 2004. 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APPENDIX B Intercept and slope coef?cients, by year, for the relationship between altitude and liver d13C values of Black-throated Blue Warblers in the Santeetlah Creek watershed (Ecological Archives A019-049-A2). APPENDIX C Intercept and slope coef?cients, by year, for the relationship between altitude and muscle d13C values of Black-throated Blue Warblers in the Santeetlah Creek watershed (Ecological Archives A019-049-A3). July 2009 1273STABLE CARBON ISOTOPES IN WOOD WARBLERS Ecological Archives: A019-049-A1 Gary R. Graves and Christopher S. Romanek. 2009. Mesoscale patterns of altitudinal tenancy in migratory wood warblers inferred from stable carbon isotopes. Ecological Applications 19:1264?1273. Appendix A. Independent analyses (ANCOVA) for ?15N values of liver and pectoral muscle of male Black-throated Blue Warblers (Dendroica caerulescens) collected during eight consecutive breeding seasons (1995-2002) in the Big Santeetlah Creek watershed. Dependent variable Independent variables df MS F-ratio P ____________________________________________________________________________ ?15N Liver Age 1 0.16 0.38 0.54 (R 2 = 0.20) Year 7 1.99 4.69 0.0001 Age X Year 7 0.30 0.71 0.67 Altitude 1 0.69 1.62 0.20 Julian date 1 0.42 1.00 0.32 Error 314 0.42 ?15N Pectoral Age 1 0.06 0.20 0.66 (R 2 = 0.26) Year 7 1.42 5.00 < 0.0001 Age X Year 7 0.45 1.57 0.14 Altitude 1 0.67 2.36 0.13 Julian date 1 1.31 4.60 0.03 Error 315 0.28 1 Ecological Archives: A019-049-A2 Gary R. Graves and Christopher S. Romanek. 2009. Mesoscale patterns of altitudinal tenancy in migratory wood warblers inferred from stable carbon isotopes. Ecological Applications 19:1264?1273. Appendix B. Intercept and slope coefficients (by year) for the relationship between altitude and ? 13Cliver values of Black-throated Blue Warblers in the Santeetlah Creek watershed (see Fig. 3). 1995 (n = 37) R 2 = 0.00 Effect Coefficient Standard error t P (two-tailed) Constant -22.32 0.59 -37.63 Altitude -0.0001 0.0005 -0.20 0.84 1996 (n = 45) R 2 = 0.00 Effect Coefficient Standard error t P (two-tailed) Constant -22.10 0.66 -33.24 Altitude -0.0002 0.0006 -0.31 0.76 1997 (n = 41) R 2 = 0.02 Effect Coefficient Standard error t P (two-tailed) Constant -23.18 0.53 -44.01 Altitude 0.0004 0.0005 0.81 0.42 1998 ( n = 33) R 2 = 0.14 Effect Coefficient Standard error t P (two-tailed) Constant -23.39 0.39 -59.42 Altitude 0.0008 0.0003 2.27 0.03 1999 (n = 37) R 2 = 0.00 Effect Coefficient Standard error t P (two-tailed) Constant -24.25 0.36 -66.85 Altitude 0.0014 0.0003 4.27 0.0001 2000 (n = 45) R 2 = 0. 00 Effect Coefficient Standard error t P (two-tailed) Constant -23.47 0.81 -64.61 Altitude -0.0001 0.0007 -0.16 0.88 2 2001 (n = 43) R 2 = 0.29 Effect Coefficient Standard error t P (two-tailed) Constant -24.82 0.28 -64.61 Altitude 0.0013 0.0003 4.04 <0.001 2002 (n = 46) R 2 = 0.01 Effect Coefficient Standard error t P (two-tailed) Constant -24.35 0.91 -26.80 Altitude 0.0006 0.0008 0.72 0.48 1 Ecological Archives: A019-049-A3 Gary R. Graves and Christopher S. Romanek. 2009. Mesoscale patterns of altitudinal tenancy in migratory wood warblers inferred from stable carbon isotopes. Ecological Applications 19:1264?1273. Appendix C. Intercept and slope coefficients (by year) for the relationship between altitude and ?13C muscle values from Black-throated Blue Warblers in the Santeetlah Creek watershed (see Fig. 3). 1995 (n = 37) R 2 = 0.05 Effect Coefficient Standard error t P (two-tailed) Constant -23.59 0.47 -50.18 Altitude 0.0006 0.0004 1.41 0.17 1996 (n = 43) R 2 = 0.002 Effect Coefficient Standard error t P (two-tailed) Constant -22.96 0.43 -53.38 Altitude 0.0001 0.0004 0.29 0.77 1997 (n = 53) R 2 = 0.17 Effect Coefficient Standard error t P (two-tailed) Constant -24.80 0.62 -40.18 Altitude 0.0015 0.0005 2.86 0.007 1998 ( n = 34) R 2 = 0.15 Effect Coefficient Standard error t P (two-tailed) Constant -24.44 0.69 -35.23 Altitude 0.0014 0.0006 2.42 0.021 1999 (n = 39) R 2 = 0.38 Effect Coefficient Standard error t P (two-tailed) Constant -24.97 0.37 -67.21 Altitude 0.0016 0.0003 4.76 < 0.0001 2000 (n = 45) R 2 = 0.14 Effect Coefficient Standard error t P (two-tailed) Constant -25.04 0.64 -39.35 Altitude 0.0015 0.0006 2.61 0.012 2 2001 (n = 44) R 2 = 0.15 Effect Coefficient Standard error t P (two-tailed) Constant -24.92 0.53 -46.74 Altitude 0.0013 0.0005 2.77 0.008 2002 (n = 46) R 2 = 0.017 Effect Coefficient Standard error t P (two-tailed) Constant -24.67 0.84 -29.26 Altitude 0.0006 0.0007 0.86 0.39