2.4 Statistical analysis
All statistical analyses were performed in R v. 3.6.1 (R Core Team,
2019). For each season, we estimated differences in
δ13C and δ15N values of western
sandpiper plasma among demographic groups using general linear models
(GLMs). To characterize the diet of western sandpipers in each season,
we first estimated differences in δ13C and
δ15N values among five groups of prey: biofilm,
microphytobenthos, Bivalvia, Polychaeta, and other invertebrates
(Clitellata, Gastropoda, and Malacostraca) using a linear discriminant
analysis (LDA). Clitellata, Gastropoda, and Malacostraca were grouped
into a single prey source because there was substantial overlap in their
δ13C and δ15N values. Statistical
significance of δ13C and δ15N values
in the LDA was determined with forward stepwise selection using Wilks’
Lambda (λ) criterion in the klaR package (Weihs, Ligges, Luebke, &
Raabe, 2005). Next, we estimated the proportional contribution of the
five prey groups to the diets of western sandpipers using a stable
isotope mixing model in the IsotopeR package (Hopkins & Ferguson,
2012). Model runs were conducted with three chains, a burn-in of 100,000
iterations, a run of 100,000 iterations, and a thinning rate of 100. We
compared the results of mixing models using discrimination factors for
plasma derived from dunlin from two previously published studies: Evans
Ogden, Hobson, & Lank (Δ 13C ± standard
deviation (SD) = 0.50 ± 0.42 ‰ , Δ 15N ±
SD = 3.30 ± 0.32 ‰ ; 2004) and Lourenço et al.
(Δ 13C ± SD = 0.32 ± 0.16 ‰ ,Δ 15N ± SD = 3.30 ± 0.20 ‰ ; 2015).
Discrimination factors were added to the stable isotope values of all
prey prior to model runs. Discrimination factors have been shown to vary
among different environments, trophic levels, taxa, tissues, metabolic
rates, modes of nitrogenous excretion, sample preparations, and food
sources with different qualities and quantities of protein (Dalerum &
Angerbjörn, 2005; Florin, Felicetti, & Robbins, 2011; McCutchan, Lewis,
Kendall, & McGrath, 2003; Robbins, Felicetti, & Florin, 2010;
Vanderklift & Ponsard, 2003). To help account for this variation, we
incorporated uncertainty in discrimination factors by including standard
deviation in our mixing models (Hopkins & Ferguson, 2012).
We compared the diet composition of western sandpipers obtained from the
mixing models between seasons, among demographic groups, and among
demographic groups within each season using a Permutational Analysis of
Variance (PERMANOVA) with 9999 permutations based on Bray-Curtis
dissimilarities in the Vegan package (Oksanen et al., 2019). We ensured
homogeneity of multivariate dispersion using permutest in Vegan (Oksanen
et al., 2019).
Finally, we examined the relationship between morphological traits of
individual western sandpipers and diet composition using redundancy
analysis (RDA). We estimated the proportion of variance in diet
composition explained by two measures of body size, body mass and bill
(culmen) length. The RDA was conducted using forward selection with
10,000 permutations using ordistep in Vegan (Oksanen et al., 2019).
Differences in body mass and bill length among demographic groups were
assessed using GLMs.
RESULTS
Western sandpiper plasma did not differ significantly among demographic
groups in δ13C (all p > 0.05) or
δ15N (all p > 0.05) in mid-winter.
In spring, δ13C (all p > 0.05)
values did not differ among demographic groups, but
δ15N values were significantly lesser in juvenile
males compared to adult females (p = 0.04). In each season,
stable isotope signatures differed among the five prey groups (Fig. 1;
Appendix Fig. A1). Only δ15N contributed significantly
to the linear discriminant function that differentiated prey groups in
winter (λ = 0.18, p < 0.001) and spring
(λ = 0.17, p < 0.001).
Discrimination factors from Evans Ogden et al. (2004) and Lourenço et
al. (2015) were similar, yielding only minor differences in mixing model
results, especially in spring (Fig. 2; Appendix Fig. A2). In winter,
results from the mixing model that used the discrimination factor of
Evans Ogden et al. (2004) indicated that western sandpipers consumed
greater proportions of biofilm, Bivalvia, and Polychaeta compared to the
results of the mixing model that used the discrimination factor of
Lourenço et al. (2015; Fig. 2; Appendix Fig. A2). We present the results
of subsequent analyses using the mixing model with the discrimination
factor of Evans Ogden et al. (2004); results using the mixing model with
the discrimination factor of Lourenço et al. (2015) are presented in
Appendix A.
The diet composition of western sandpipers estimated by the mixing model
differed significantly between seasons (pseudo-F = 3040.86,R2 = 0.95, p < 0.001), among
demographic groups (pseudo-F = 37.04, R2= 0.01, p < 0.001), and among demographic groups within
each season (pseudo-F = 26.15, R2 =
0.01, p < 0.001; Appendix Table A2). The difference
between seasons explained 95% of the variation in western sandpiper
diets, whereas the differences among demographic groups and among
demographic groups within each season each explained 1% of the
variation in diet. The proportions of biofilm and Bivalvia in western
sandpiper diets were similar between seasons (Fig. 2). Western
sandpipers from all demographic groups consumed a large proportion of
other invertebrates in winter, whereas they consumed greater proportions
of Polychaeta and microphytobenthos in spring (Fig. 2). However, the
shift from diets dominated by other invertebrates in winter to diets
dominated by Polychaeta in spring observed in all demographic groups
should be interpreted with caution; this shift could be an artifact of
the substantial overlap in stable isotope values between other
invertebrates and Polychaeta in spring (Fig. 1). In contrast, the stable
isotope values of microphytobenthos had minimal overlap with other prey
groups during both seasons (Fig. 1). In winter, the average consumption
of biofilm and microphytobenthos combined was approximately 2% of the
diet of western sandpipers regardless of demographic group, whereas in
spring, biofilm and microphytobenthos comprised 9%, 13%, 19%, and
24% of the diets of adult females, adult males, juvenile females, and
juvenile males, respectively. Some juveniles consumed a large proportion
of microphytobenthos in spring (Figs. 2-3). The mixing model indicated
that the combined contribution of biofilm and microphytobenthos made up
greater than 25% of the diet in 13 of 70 (19%) western sandpipers
captured in spring; of these 13 individuals, all were juveniles, and 9
(69%) were juvenile males (Fig. 2).
In winter, there was little variation in prey consumption among
demographic groups (Fig. 2); therefore, we did not analyze these data
with an RDA. In contrast, diet composition differed among demographic
groups in spring (Fig. 3; Appendix Fig. A3). Bill length and body mass
were both selected as significant variables that explained differences
in spring diet composition among western sandpipers (Fig. 3). Together,
these variables explained 31% of the total variation
(R2adj = 0.31). Bill length and
body mass were significantly greater in female western sandpipers
compared to males (all p < 0.05), but, within each sex,
morphometric measurements did not differ significantly between age
classes (all p > 0.05). Males with lesser body mass
consumed a greater proportion of other invertebrates compared to females
(Fig. 3). Consumption of Polychaeta increased with increasing bill
length and was greatest in adult females (Fig. 3). In contrast,
consumption of microphytobenthos increased with decreasing bill length
and was greater in juveniles, particularly juvenile males (Fig. 3).
DISCUSSION
Our results provide the first
evidence of seasonal differences in diet composition among demographic
groups of western sandpipers, strongly supporting the conclusion that
age- and sex-related dietary specialization facilitate seasonal resource
partitioning in this species. In spring, juveniles, particularly
juvenile males, consumed more biofilm and microphytobenthos than adults
supporting our hypothesis that sandpipers with shorter bills, lesser
mass, and less prey handling experience would consume the greatest
amount of biofilm and microphytobenthos. Further, differences in diet
composition among demographic groups were more pronounced at the onset
of migration in spring compared to mid-winter following the expectation
that the consumption of biofilm and microphytobenthos provides energy
for sandpipers during their breeding migration and reduces competition
when high densities of birds occupy foraging sites.