PCA plot—Multivariate analysis of stoichiometric relations in honeybee castes and sexes.

<p>The first two axes are presented. Queens are separated from the other castes/sexes primarily due to their relatively high concentrations of Zn, Na, Fe, Mg, Ca, Mn and S. Drones form a cluster separate from all females mainly due to the males’ relatively high concentrations of C and N and low concentrations of P, Ca, Mg and Zn. These tendencies were confirmed by ANOVA computed independently for the 1^st and 2^nd axis scores (p<0.05).</p

Average concentrations of elements in honeybee castes and sexes.

<p>Average concentrations of elements in honeybee castes and sexes.</p

Concentrations of elements in honeybee pollen pellets (Means and CI).

<p>ANOVA, p<0.05. Various lowercase letters denote statistically significant differences. Mixed pollen pellets (polyfloral, as they were gathered by bees) are shown in blue and sorted pellets (sorted from mixed pollen pellets, which had varying compositions of pollen from different species but contributed to the compositions of mixed pollen pellets) are shown in gray. Commercial pollen pellets: mixed, 1; sorted, 1A to 1C. Study site pollen pellets: mixed, 2; sorted, 2A to 2D. See text for explanation. Whiskers denote 95% confidence intervals. Detailed results are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183236#pone.0183236.s004" target="_blank">S4 Table</a>. Sorted pollen pellets exhibited greater variance in the element concentrations; mixed pollen pellets tended to have average concentrations of the studied elements.</p

Mineral composition of nectar stored in beehives.

<p>Mean values were calculated for 24 samples collected from 5 beehives.</p

Limiting effects on honeybee growth and development by stoichiometry of pollen from various taxa.

<p>Calculated as <i>TSR</i> ratios (<i>TSR</i> values ≥4 denote limitations on growth and development) based on published elemental composition data on pollen from various taxa and the data from this study on C contents of pollen and the elemental composition of honeybees (mean values). The effects are caste- and sex-dependent and are indicated by symbols: Q–queen, W–worker, D–Drone; “-” no limiting effect; n.d.–no data. The limiting effect of Fe calculated in the study by Todd and Bretherick [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183236#pone.0183236.ref050" target="_blank">50</a>] is probably overestimated, as indicated by italics and “(?)”. Shaded and bolded font denotes pollen taxa that are stoichiometrically well balanced for the honeybee and shaded and underlined font indicates highly stoichiometrically unbalanced pollen taxa.</p

Main (>16%) taxa composing the studied <i>PPMs</i>.

<p>Bold text indicates pollen with more than 70% of the contents obtained from a single species. Detailed data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183236#pone.0183236.s003" target="_blank">S3 Table</a>. Mixed <i>PPMs</i> 1 and 2 constituted the pool of pollen that was collected by the honeybees. Sorted <i>PPMs</i> (1A-C sorted from 1 and 2A-D sorted from 2) had specific species compositions that differed from those of mixed <i>PPMs</i> but contributed to those of mixed <i>PPMs</i>.</p

Variability, ranges and mean values of elemental concentrations in pollen of various taxa (one species, one genus, one family, polyfloral).

<p>Information is based on data in the literature collected worldwide.</p

Average total amounts of elements in honeybee castes and sexes.

<p>Average total amounts of elements in honeybee castes and sexes.</p

Symbolic representation of the process of pollen sorting from mixed to sorted <i>PPMs</i>.

<p>Symbolic representation of the process of pollen sorting from mixed to sorted <i>PPMs</i>.</p

Trophic stoichiometric ratios (<i>TSR = (C</i>:<i>X)Pollen Pellets/(C</i>:<i>X)Bee</i>; <i>C</i> = Carbon Concentration, <i>X</i> = Concentration of Element <i>x</i>) for the studied elements.

<p><b>Possibly limiting and non-limiting elements, <i>PPM</i>2.</b> TSR values ≥4 denote limitations on growth and development. The dotted red line indicates the threshold value of TSR = 4. Limitation symbols (<b>1, 2, 3, -</b>) denote the possibility of a limitation of bee growth and development due to the scarcity of a certain element in pollen. Calculated <i>TSR</i> values were grouped into 4 categories: (<b>1</b>) limiting–more than 75% of calculated <i>TSR</i> values ≥4; (<b>2</b>) likely limiting– 50–75% of calculated <i>TSR</i> values ≥4; (<b>3</b>) possibly limiting– 25–49% of calculated <i>TSR</i> values ≥4; (-) non-limiting–more than 75% of calculated <i>TSR</i> values <4. <i>PPM</i> = pollen pellet morphospecies. For every element and caste, <i>PPMs</i> are placed with 1 at the farthest left, followed by 1A-C, 2 and 2A-D (farthest right). Mixed <i>PPMs</i> 1 and 2 = pollen pellets gathered by honeybees; sorted <i>PPMs</i> = pollen pellets of different pollen species composition that were sorted from the mixed <i>PPMs</i>. <i>PPMs</i> categories are explained in the text. For each element, considered separately, various lowercase letters denote statistically significant differences (indicated as “differences” in the figure). Kruskal-Wallis test, p<0.001; for every single element: for <i>PPM</i>1 12 grouping variables, N = 36000, for PPM2 15 grouping variables, N = 45000. See text for an explanation of the <i>TSR</i> calculation and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183236#pone.0183236.s005" target="_blank">S5 Table</a> for detailed results. All of the <i>PPMs</i> limit bee development due to Na scarcity. Concerning other elements, mixed <i>PPMs</i> tend not to be limiting for bees, while sorted <i>PPMs</i> tend to be limiting to varying degrees. The possibility of co-limitation by scarcities of S, Cu, P, K, Zn or N depends on <i>PPM</i>, species composition, and caste/sex.</p