Temporal variation in resource availability, amplified by global change, may have strong impacts on species breeding at temperate and high latitudes that cue their reproduction to exploit seasonal resource pulses. This study examines how resource availability and parental care influence niche partitioning between and within age classes in the rufous-collared sparrow, which provides extensive parental care. We hypothesized juveniles would exhibit narrower niches focused on high-quality resources compared to adults, regardless of resource availability. We used stable isotope analysis to quantify individual and population niches in juveniles and adults across the breeding season in two cohorts experiencing contrasting resource landscapes. Contrary to our initial hypothesis, juveniles exhibited greater among individual diet variation and smaller total niche widths (i.e., higher levels of individual specialization) during periods of high food availability in comparison to periods of food scarcity. Interestingly, total niche width and individual specialization of adults remained stable across seasons despite a shift in trophic level, highlighting their potential role in providing a consistent diet for their young. These findings reveal a dynamic interplay between resource availability, parental care, and individual specialization, with important implications for understanding population resilience under variable resource scenarios. The study also suggests that adult sparrows modify their provisioning strategies based on resources, potentially buffering offspring from environmental fluctuations. Understanding age-specific responses to resource variation is crucial for predicting species responses to ecological conditions, particularly in regions like central Chile where seasonal resource limitation is expected to become more variable in response to climate change.

Fieldwork and Tissue Collection. The study site, Quebrada de la Plata (33°31’S 70°50’W) in central Chile, is characterized by a Mediterranean climate with cold wet winters and relatively hot dry summers (Di Castri and Hajek, 1976). The average accumulated winter precipitation is 230 mm, and during the breeding season in our study, it was 180 mm (CR2, 2024). In addition, at this site the availability of insects and plants varies seasonally being highest in spring and lowest in winter; summer values also are lower than in spring (Lopez-Calleja, 1995). We captured rufous-collared sparrows with mist nets in spring (November 2020) and summer (January 2021). We categorized captured individuals into adults (N_spring= 16, N_summer= 20) or juveniles (fledglings; N_spring= 12, N_summer= 8) according to plumage characteristics described in Miller and Miller (1968). We ringed the captured birds to ensure the independence of our samples, avoiding the resampling of the same individuals across seasons.

Parental care in this species extends from hatching until complete independence at 40 days old, with both parents actively feeding the chicks. Fledglings leave the nest after 18-20 days; at which time they start learning to eat on their own and can feed independently from four weeks old. Thus, they continue to solicit and receive food from their parents until about 40 days old, when they become completely independent (Miller and Miller, 1968; Tubaro, 1990). Accordingly, individuals captured during November (spring) would correspond to late September broods, while those captured in mid-January (summer) would correspond to early December broods, ensuring that both broods were raised under markedly different ecological conditions.

Immediately after capture, individuals were placed in a bird-holding bag (Ecotone, Poland), weighed to the nearest 0.1 g on a digital scale, and claw tips were sampled and placed in an Eppendorf tube. We then collected ~50μl blood from the brachial vein using heparinized microhematocrit capillary tubes. Blood was centrifuged at 10,000 rpm for 10 min to separate plasma and red blood cells. All samples were stored frozen in liquid nitrogen prior to preparation for stable isotope analysis. All tissue samples were dried at 80°C, homogenized, and ~0.5–0.6 mg of each sample was loaded into tin capsules for stable isotope analysis.

Resource Availability. The rufous-collared sparrow is an omnivorous species that routinely consumes arthropods, seeds, and small herbaceous plants (Lopez-Calleja, 1990, 1995). In order to support the information given by previous research by Lopez-Calleja (1995) at the study site, we examined seasonal variation in resource availability measured as the abundance and diversity of herbaceous plants and terrestrial arthropods. Herbaceous plants were sampled in five permanent 1m² quadrats distributed across the study area which covered approximately 1000 m² within a ravine, ensuring each quadrat was positioned at a minimum distance of 35 meters apart. The greatest distance between two quadrats was 140 meters. On each quadrat, we identified the percent cover of living herbaceous species during both seasons: mid-September and mid-October for spring and mid-December and mid-January for summer. Also, we sampled terrestrial arthropods with Barber pitfall traps filled with water and biodegradable detergent (Southwick, 1978). They were placed every 1 m along five linear transects of 10 m distributed across the study area, at close proximity from the vegetation quadrats, and collected after 24 h (Jaksic and Lazo, 1999). We counted and identified the collected arthropods and classified them into morphotypes corresponding to the lowest taxonomic resolution possible. We then estimated the mean total abundance per sample unit for herbaceous plants and terrestrial arthropods for each season and its confidence interval by bootstrap procedure with 10,000 iterations.

Resource diversity was estimated by quantifying the Hill numbers for the plant and arthropod assemblages (Chao et al., 2014). Seasonal species richness and Shannon diversity index were calculated by using sample-size-based rarefaction and extrapolation estimation of the Hill numbers of order q equal to 0 and 1, respectively. The estimates were based on species abundance, measured as the percent cover of living plant species and number of individuals within each arthropod morphotype (Chao et al., 2014; Hseih et al., 2019). All estimations were performed with the package iNEXT (Hseih et al., 2019).

Moreover, we collected leaf and seed samples from 12 plant species during the spring and seed samples from 6 species during the summer, as live plant cover was absent in our study quadrats. The plant and seed species were selected based on their relative abundance and their inclusion in the rufous-collared sparrow's diet as described by Lopez-Calleja (1990,1995). This collection allowed us to characterize the d¹⁵N composition of the trophic web baseline required to estimate relative trophic level (see below).

Stable Isotope Analysis. We measured d¹⁵N values in blood plasma, red blood cells, and claw tips of juvenile and adult individuals to quantify niche characteristics in seasons characterized by high (spring) and low (summer) resource availability according to previous studies (Lopez-Calleja, 1995). Although many tissues can be compared to construct a time series of isotopic variation, blood plasma, red blood cells, and claws can be easily collected in the field without sacrificing animals (Podlesak et al., 2005; Carleton et al., 2008). d¹⁵N values of claw tips integrate dietary inputs over ~6 weeks prior to sample collection, while blood plasma and red blood cells integrate diet over ~1-2 and ~3-4 weeks prior to collection, respectively (Hobson and Clark, 1992; Carleton et al., 2008; Hahn et al., 2014). Young birds initiate the post-juvenile molt either at the time of independence or within about 10 days thereafter (Miller and Miller, 1968). In our study, the captured juveniles showed no evidence of molting, which suggest that they probably were not yet fully independent in their feeding. This implies that the variance in diet composition based on analysis of these three tissues, with the most recent one reflecting diet integrated over ~1-2 weeks prior to capture, represents mostly the diet of juvenile individuals during the parental care period when they had not yet achieved foraging independence.

Nitrogen isotope (d¹⁵N) values of sparrow tissues were measured on a Costech 4010 elemental analyzer coupled to a Thermo Finnigan Delta Plus XP isotope ratio mass spectrometer at the University of New Mexico Center for Stable Isotopes (Albuquerque, NM). Stable isotope data are expressed in delta (d) notation as parts per thousand (‰) according to the equation, d¹⁵N = (R_sample / R_standard – 1) X 1000, where R represents the ratio heavy to light isotopes (¹⁵N/¹⁴N) of the sample relative to the international reference standard. Measured d¹⁵N values were calibrated relative to air with certified reference materials. Within-run estimates of analytical precision were obtained by measurements of an internal laboratory reference material and yielded a precision (SD) ≤ 0.2‰.

Total Niche Width and Individual Diet Specialization. To quantify the total niche width and degree of individual diet specialization between age groups and seasons, we used a modified version of Roughgarden index for use with d¹⁵N data (Roughgarden, 1972; Maldonado et al., 2017, 2019). Roughgarden used a simple framework to quantify individual diet specialization: the total niche width (TNW) of the (adult or juvenile) age group is the sum of two components: (1) the within-individual component (WIC) which reflects the average of the variability of resources used by individuals, and (2) the between individual component (BIC) that represents the between-individual variation in the average use of resources. The degree of individual diet specialization is reflected in the WIC/TNW ratio where low ratios close to zero indicate a higher prevalence of specialization within an age class, indicating a generalist population composed of individual specialists that each use a small subset of the total niche width.

Trophic Level. In food web studies involving animals, d¹⁵N measurements are commonly used to infer the relative trophic position of consumers due to the predictable and significant enrichment of ¹⁵N at higher trophic levels, known as trophic enrichment factors (TEFs). This enrichment occurs because of protein catabolism. During catabolism, amino acids with amine groups containing the heavier ¹⁵N isotope are preferentially retained over those with the lighter ¹⁴N isotope. Consequently, excreted urinary nitrogen is depleted in ¹⁵N compared to the animal's tissues. This leads to a progressive accumulation of ¹⁵N along the food chain, with each successive trophic level exhibiting higher ¹⁵N/¹⁴N ratios than the previous level (Post, 2002; see also Karasov & Martinez del Rio, 2007 and references therein). Additionally, using stable isotopes to estimate trophic levels requires a priori estimates of discrimination factors, which represents the differences in isotopic composition between an animal's tissue and its diet. These factors are crucial for accurately interpreting d¹⁵N data and inferring trophic positions in bird studies, as they vary depending on the tissue being analyzed. We used previously determined discrimination factors for bird tissues in our trophic level calculations (Caut et al.2009). We calculated the relative trophic level (TL) of each individual as follows (Post, 2002): TL= (1 + (d¹⁵N_bird − d¹⁵N_producers) / Δ¹⁵N, where the d¹⁵N_bird represents the mean isotopic signature of tissues, d¹⁵N_producers correspond to the baseline isotopic signature of the primary producers (plants) and Δ¹⁵N is the trophic discrimination factor. We used a trophic discrimination of 2.37‰ for collagen (claws), 2.25‰ for blood, and 2.82‰ for plasma (Caut et al., 2009). To calculate the baseline, we estimated the d¹⁵N values of plants and seeds for both seasons, but a t-test showed no significant differences between them. Therefore, we used a pooled sample d¹⁵N value of 3.47‰ for the baseline at the study site.

Statistical Analysis. To assess for differences in levels of IS of juveniles and adults against a null model, we employed a non-parametric Monte Carlo procedure with 10,000 iterations to derive p-values. The null model characterizes the total niche width of either juveniles or adults as being comprised of generalist individuals who forage randomly across the entire niche of their respective age classes (Bolnick et al., 2002). This Monte Carlo procedure is implemented in the R package RInSp (Zaccarelli et al., 2013). To evaluate whether significant differences exist in TNW, BIC, WIC, and WIC/TNW between juveniles and adults, we also used a Monte Carlo permutation procedure. To calculate a p-value, the observed mean difference in WIC/TNW between juveniles and adults was compared against the mean differences generated from 10,000 random permutations of juvenile-adult groups. We then assessed potential differences in trophic levels between the two age classes across seasons by linear mixed models (lmm; package lme4, Bates et al., 2015) with age class (juveniles, adults), seasons (spring, summer) and the interaction among them as fixed factors and tissue (nails, blood plasma and red blood cells) as random intercept. Model assumptions (normality, homoscedasticity and absence of residual autocorrelation) were confirmed. Finally, we estimated the isotopic niche overlap between juveniles and adults in both seasons by calculating the overlapped areas (and their bootstrap standard errors with 1000 replicates, SE) relative to each pair of d¹⁵N kernel density estimations using the overlapping R package (Pastore, 2018; Pastore and Calcagnì, 2019). To assess whether the estimated area of overlap was significantly different from what would be expected by chance -due solely to sample size- we performed a permutation test with 10,000 random permutations of the d¹⁵N values. We then compared the observed overlap area to the 95% confidence interval (CI_0.95) generated from the permuted data.