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3 Nest material preferences by spotless starlings
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* 5 Cristina Ruiz-Castellano , Gustavo Tomás, Magdalena Ruiz-Rodríguez and
6 Juan J. Soler
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9 Departamento de Ecología Funcional y Evolutiva, Estación Experimental de Zonas Áridas
10 (EEZA-CSIC), Ctra. Sacramento s/n, La Cañada de San Urbano, E-04120 Almería, Spain.
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12 * For correspondence: e-mail: [email protected], Tf: +34 950281045 (Ext: 784)
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14 Abstract
15 The avian nest is an essential structure for offspring development. For adults, nest building
16 entails costs in terms of time, energy and exposure to predators and parasites. Amount and
17 diversity of materials used for nest building depend on their availability and functionality in
18 scenarios of sexual selection and parasitism. Green plants and feathers of different colors
19 have been hypothesized to play key roles in offspring protection against pathogens, and we
20 here experimentally assessed spotless starling (Sturnus unicolor) preferences for pigmented
21 vs. unpigmented feathers and for different green plants (aromatic vs. non-aromatic plants)
22 as nest materials. We predicted a preferential selection of unpigmented feathers and
23 aromatic plants according to the antimicrobial properties of these materials described in the
24 literature. We evaluated these predictions during nest building and during egg-laying
25 stages. As expected, starlings preferentially selected unpigmented feathers both before and
26 during egg laying, while aromatic plants were preferentially selected only during the egg-
27 laying stage. These results suggest that starlings prefer nest materials that enhance
28 antimicrobial protection of their offspring. We also discuss some other, non-exclusive
29 functions that might explain the observed preference for nest materials, especially with
30 regard to their potential role in sexual signaling.
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32 Keywords: Aromatic plants, Feather pigmentation, Nest building behavior, Nest lining
33 feathers, Nest material preference.
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35 Introduction
36 The avian nest is an essential structure that protects offspring from a wide diversity of
37 environmental challenges during development (Hansell 2000; Moreno 2012; Mainwaring et
38 al. 2014). Nest building entails costs in terms of, for instance, expended time and energy, or
39 exposure to predators and parasites (Hansell 2000; Mainwaring and Hartley 2013).
40 Different nest materials have different properties, so birds are expected to carefully adjust
41 nest-building behavior to optimize the balance between costs and benefits while selecting
42 the more appropriate combination of materials to maximize fitness (Soler et al. 1998;
43 Hansell 2000).
44 Among the several materials employed by birds to build their nests, the use of green
45 plants and feathers has received special attention in evolutionary ecology research (Hansell
46 2000; Dubiec et al. 2013). Green plants, which are used as structural or lining material, may
47 also have several non-exclusive functions in avian nests that include sexual signaling
48 (Fauth et al. 1991; Brouwer and Komdeur 2004; Polo et al. 2004; Veiga et al. 2006;
49 Moreno 2012; Tomás et al. 2013). Yet, the most studied function of green plants is related
50 to the antimicrobial and antiparasitic properties of their volatile secondary compounds and
51 essential oils that protect offspring from pathogens (Clark and Mason 1985; Lafuma et al.
52 2001; Gwinner and Berger 2005; Shutler and Campbell 2007; Mennerat et al. 2009a;
53 Dubiec et al. 2013; Scott-Baumann and Morgan 2015). Through different metabolic routes,
54 many of these plants can also have therapeutic effects improving nestling growth, health, or
55 immunocompetence (Gwinner et al. 2000; Gwinner and Berger 2005; Mennerat et al.
56 2009b). Therefore, the use of green plants in the nests of birds is considered as a form of
57 self-medication (De Roode et al. 2013). Different plants have different compounds, so birds
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58 may adjust their nest building behavior according to the selective pressures exerted by
59 pathogens.
60 Feathers are another material commonly used in bird nests, mainly for nest cup
61 lining, but also as structural material (Hansell 2000). Although the most advocated function
62 of feathers in nests is related to thermal insulation (Møller 1984; Lombardo et al. 1995;
63 Hilton et al. 2004; Pinowski et al. 2006; Dawson et al. 2011; Windsor et al. 2013), they can
64 also be involved in sexual signaling (Polo and Veiga 2006; Sanz and García-Navas 2011;
65 García-López de Hierro et al. 2013; Mainwaring et al. 2016) and offspring protection from
66 microbial infections (Soler et al. 2010). These two last functions are respectively based on
67 attractiveness of feathers of different color (Veiga and Polo 2005; Avilés et al. 2010, Sanz
68 and García-Navas 2011), and on the antimicrobial properties of the bacterial communities
69 that grow digesting the keratin of feathers (Peralta-Sánchez et al. 2014; Ruiz-Castellano et
70 al. 2016). These two functions are not mutually exclusive, and we here concentrate our
71 predictions on the hypothetical antimicrobial functionality of feathers. Apparently, bacterial
72 growth (Goldstein et al. 2004; Gunderson et al. 2008) and antimicrobial properties are
73 higher for bacterial communities of unpigmented feathers (Peralta-Sánchez et al. 2014).
74 Thus, birds may preferentially select unpigmented feathers for nest building.
75 Some of these hypothetical effects of feathers and green plants in the nests of birds
76 have been experimentally demonstrated, but evidence of associated adaptive preference is
77 restricted to a few species. For green plants, previous research on European starlings
78 (Sturnus vulgaris) has suggested olfactory discrimination of plant volatiles (Clark and
79 Mason 1987; Gwinner and Berger 2008). Moreover, an observational study concluded that
80 plant species composition of blue tit (Cyanistes caeruleus) nests results from individual
81 preferences of particular species (Mennerat et al. 2009c). With respect to feathers, Peralta-
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82 Sánchez et al. (2011) found that barn swallows (Hirundo rustica) preferentially selected
83 white feathers (experimentally offered) for lining their nests. Exploring these preferences in
84 some other species, as well as possible variation depending on environmental conditions,
85 would aid understanding of the functional significance of nests and nest building behaviors.
86 Selection of nest materials by birds can be tackled by looking at the final
87 assemblage of materials in nests after considering the availability of different materials in
88 the nest surroundings (Petit et al. 2002; Mennerat et al. 2009c; Pires et al. 2012). However,
89 birds could also collect nest materials far away from their territories (Bailey et al. 2016),
90 which challenges a proper statistical control of nest material availability. Therefore, an
91 experimental approach might be the most suitable way to study the preference for certain
92 nest materials, which we accomplished here by manipulating their availability. During nest
93 building stage, unlimited pigmented and unpigmented feathers, as well as different
94 aromatic and non-aromatic plant species, were offered to spotless starlings. Later we
95 recorded its presence and abundance in their nests. We predict that birds would select
96 preferentially unpigmented feathers and aromatic plants if antimicrobial protection of the
97 offspring were one of the functions of these nest materials.
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99 Materials and Methods
100 Study species and area
101 The spotless starling is a medium-sized, hole-nesting passerine that mostly breeds in
102 colonies (Cramp 1998). Green plants and feathers are commonly used as nest materials,
103 with plants and feathers being carried to the nest by males and females respectively (Polo
104 and Veiga 2006). Both materials are embedded in the nests, forming part of both their
105 structural and lining layers (Veiga and Polo 2016). Starlings in our population usually
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106 commence to build their nests in March, laying eggs at mid-April. Since April 10th nest-
107 boxes were visited every three days until the first egg was laid.
108 The study was performed during the 2013 breeding season in a population located
109 in the old railway station of La Calahorra (37°18’ N, 3°11’ W) within the Hoya de Guadix,
110 a high-altitude plateau 1000 m a.s.l., with a semi-arid climate. Apart from a few sparse
111 almond trees (Prunus dulcis), vegetation in the area is typical of an agro-pastoral steppe,
112 with abandoned fields interspersed within barley (Hordeum vulgare) and lettuce (Lactuca
113 sativa) cultivated fields. There were 80 cork-made nest-boxes (internal height * width *
114 depth: 350 * 180 * 210 mm, bottom-to-hole height: 240 mm) available for spotless
115 starlings, attached to tree trunks or walls at 3-4 m above ground. Nest-boxes were often less
116 than 1 m apart from each other (as in natural holes).
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118 Experimental procedures
119 Feathers and plants were offered ad libitum to starlings during nest building and laying
120 stages. Feathers were provided in 24 * 24 cm broad plastic meshes placed in the ground,
121 and distributed around the study area, so that all breeding pairs could have access to
122 feathers. Twenty meshes were located in adjacent pairs, containing 50 unpigmented and 50
123 pigmented feathers each. Feathers offered to starlings, of approximately 10 cm long, were
124 from domestic turkeys (Meleagris gallopavo) to distinguish them from chicken feathers
125 that were the most abundant in starling nests in our population. In addition, turkey feathers
126 were marked on the quill with a permanent marker. Meshes were checked daily and
127 feathers were replenished whenever there were 15 feathers or less in the mesh.
128 Plants were provided in plastic containers (height * width * length: 6 * 7 * 9.5 cm)
129 filled with insulating foam and water to maintain them fresh. In each container, four apical
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130 plant fragments of approximately 10 cm length of four aromatic species (Marrubium
131 vulgare, Artemisia barrelieri, Lamium amplexicaule and Anacyclus clavatus) and one non-
132 aromatic plant (barley, Hordeum vulgare) were provided. Plants were marked in the stem
133 with seal ink. Containers were attached on top of 2/3 of nest-boxes and were replaced every
134 three days to provide fresh plants, when the number of fragments of each plant species
135 collected by starlings was counted. Starlings made use of plants irrespective of whether
136 those were above their own or above adjacent nest-boxes (authors’ personal observation)
137 and, if considering six consecutive containers, plant fragments of all offered species were
138 always available at the time of container replacement.
139 Starting one week before the expected laying date of the first egg (the onset of egg
140 laying of starling pairs in our population is quite synchronous), plants and feathers were
141 provided to starlings during three weeks. Nest-boxes were checked every three days, from
142 the day plants and feathers were offered until eggs were found in the nest. During each
143 visit, the number of feathers present in nests was counted, distinguishing between
144 pigmented and unpigmented, and between experimental and non-experimental feathers.
145 Plants present in nests were also weighted (± 0.1 g) and fragments of experimental plants
146 found in nests were counted. When experimental nest materials were offered for the first
147 time, none of the nests contained eggs. Some nests (N = 21) were detected with eggs three
148 days later, during the second visit, while for 27 nests, eggs were detected in subsequent
149 visits. Since nest material composition during the pre-laying stage was estimated during the
150 visit just before eggs were detected, only that of the latter group of nests could have been
151 influenced by the experimental ad libitum availability of materials. Thus, these two groups
152 of nests serve to test for the influence of the experiment on nest material composition
153 before egg laying.
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154 In addition, as proxies of the selection made by starlings of feathers and plants, the
155 number of times that each experimental mesh containing either pigmented or unpigmented
156 feathers needed to be replenished, and the number of steams of each plant species that had
157 been collected from the containers, were used.
158 To estimate plant availability around nests, the percentage of a circular area of 20
159 m-radius around each nest-box that was covered by green plants was recorded (hereafter
160 percentage of vegetation coverage). These values were used for statistically controlling
161 plant availability when analyzing variation in mass of green plants found in starling nests.
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163 Antimicrobial activity of green plants offered to starlings
164 The antimicrobial activity of the five green plants offered to spotless starlings was assessed
165 in the laboratory. First, plants were sterilized using a UV sterilizer chamber (Burdinola BV-
166 100). Second, 1 cm fragments of each species were placed in Brain Heart Agar (BHA,
167 Scharlau Chemie S.A., Barcelona) plates, inoculated with 100 µL of a 24 h culture of the
168 indicator bacteria strain (see below), and then incubated 24 h at 37 ºC. The antimicrobial
169 activity of each plant was revealed by the presence of growth-inhibition halos around the
170 plant fragment. Two kinds of halos were distinguished: a clear halo, where growth of the
171 indicator bacteria was completely inhibited, and a colored halo, presumably due to
172 pigments from plants that do not completely inhibit growth of the indicator bacteria.
173 Antimicrobial activity of each plant was summarized by an index resulting from the
174 addition of standardized values (i.e., mean = 0 and SD = 1) of three different variables
175 describing (i) halo transparency: 0 (no halo), 1 (not clear halo), 2 (clear halo); (ii) size of
176 halo: 0 (no halo), 1 (1-2 mm), 2 (3-4 mm), 3 (5-6 mm) and 4 (>6 mm), and (iii) number of
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177 indicator bacteria to which the tested plant demonstrated antimicrobial activity (from 0 to
178 11).
179 Antimicrobial activity assays were performed against 11 bacterial typified strains
180 from the Spanish Type Culture Collection (CECT) and from our own laboratory collection:
181 Bacillus licheniformis D13, Enterococcus faecalis MRR10, Listeria inocua CECT340,
182 Listeria monocytogenes CECT4032, Staphylococcus aureus CECT240, Enterococcus
183 faecalis F-58, Enterococcus faecalis JH2, Enterococcus faecalis S47, Enterococcus
184 faecium 115, Lactobacillus paracasei 11-2 and Lactococcus lactis. However, it should be
185 noted that none of the tested plant species showed antimicrobial activity against the last six
186 indicator bacteria.
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188 Sample size and statistical analyses
189 We used information on feathers and plants in 48 nests at the two different times of interest:
190 once eggs were found in the nest for the first time (hereafter laying stage), and in the
191 previous visit three days before (hereafter pre-laying stage). Total and experimental number
192 of pigmented and unpigmented feathers, as well as mass of plants found in starling nests,
193 were log10 transformed to approach Gaussian distributions. Thus, transformed variables
194 and/or the residuals of the statistical models did follow normal distributions.
195 Due to variability in laying dates, experimental feathers and plants were only
196 available for 27 out of the 48 nests sampled during the pre-laying stage, but for all of them
197 during the laying stage. Thus, to explore differences in number and color composition of
198 feathers in nests due to the presence of ad libitum experimental feathers, we performed two
199 different models. First, we explored the effects of availability of feathers (nests with vs
200 without experimental feathers available, fixed factor) and feather pigmentation on number
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201 of feathers detected in nests during the pre-laying stage (dependent variable). We used a
202 Repeated Measures ANOVA (RM-ANOVA) with numbers of feathers of each color
203 detected in the same nest as within effects, and availability of experimental feathers as
204 between factor. Furthermore, to explore the effects of reproductive stage (i.e., pre-laying vs
205 laying) and feather composition on total number of feathers in starling nests, we only used
206 nests that had experimental feathers available both before and during egg-laying
207 reproductive stages. These effects were explored in a RM-ANOVA with two within factors,
208 feather color and reproductive stage. The effects of laying date on repeated measures (i.e.,
209 interactions) were estimated in separate models. In addition, to analyze feather color
210 preference by starlings, we used the number of times that meshes of different color were
211 replenished as dependent variable, and feather color as fixed factor in a GLM.
212 The effect of plant availability on the amount of green plants found in starling nests
213 was explored in GLM models with availability of experimental plants as the categorical
214 factor, and the percentage of vegetation coverage as a covariable. The effect of nest stage
215 on the amount of green plants in starling nests was explored using nests with available
216 experimental green material during the pre-laying and laying stages. For this, we used a
217 RM-ANOVA with nest stage as repeated measures variable. The effect of laying date on
218 the repeated measures variable (i.e., interactions) was estimated in a separate RM-ANOVA.
219 In addition, we further explored the preference of starlings for any of the experimental
220 plants offered in a Generalized Linear Model (GLZ) with a binomial distribution and logit-
221 link function while controlling for overdispersion. We used the presence/absence
222 information of experimental plants of each species in starling nests (during the pre-laying
223 and laying stages) as dependent variable, and plant species and nesting stage as categorical
224 independent factors. Plant preferences were also assessed by analyzing number of plants of
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225 each species that were collected by starlings from the containers. We used the log10
226 number of steams collected by starlings as dependent variable, and species and container
227 identity as factors in a GLM.
228 FDR (false discovery rate) procedure was used to adjust P values for multiple
229 comparisons by p.adjust function of stats package in R 3.4.1 (http://www.r-project.org/).
230 All other statistical analyses were performed with Statistica 8.0 (Statsoft Inc. 2011). Values
231 reported are means ± 95% CI.
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233 Results
234 Feather color preferences
235 During the pre-laying stage, unpigmented feathers were significantly more abundant in
236 starling nests than pigmented feathers (F1,46 = 6.73, P = 0.029). Moreover, neither number
237 of feathers (F1,46 = 0.41, P = 0.603), nor feather color composition (F1,46 = 0.04, P = 0.604;
238 Fig 1A) depended on availability of experimental feathers before egg laying started.
239 Nest stage (laying vs pre-laying) affected total number of feathers in nests. Feathers
240 were more abundant in starling nests at the time of laying (mean (95% CI) = 9.37 (6.27 –
241 12.47)) than before egg-laying began (mean (95% CI) = 4.89 (2.50 – 7.28)) (F1,26 = 21.84,
242 P < 0.001), with laying date explaining a non-significant proportion of variance (F1,25 =
243 0.57, P = 0.399). In addition, number of feathers in starling nests depended on feather color.
244 Unpigmented feathers (mean (95% CI) = 9.93 ± (6.69 – 13.16)) were significantly more
245 abundant than pigmented feathers (mean (95% CI) = 4.33 (1.19 – 7.18)) (F1,26 = 18.00, P <
246 0.001), and this difference tended to be more pronounced during the laying stage (F1,26 =
247 4.73, P = 0.071, Fig 1B). Laying date affected differences in feather color composition
248 since unpigmented feathers were relatively more abundant than pigmented feathers as the
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249 season progressed (F1,25 = 4.47, P = 0.071), but did not affect the detected effects of the
250 interaction between feather pigmentation and reproductive stage (F1,25 = 0.12, P = 0.737).
251 Similar results were achieved when only experimental feathers found in nests were
252 considered. Unpigmented experimental feathers were significantly more abundant (mean
253 (95% CI) = 4.81 (2.94 – 6.69)) than experimental pigmented feathers (mean (95% CI) =
254 0.02 (-0.01 – 0.38)) (F1,25 = 48.12, P < 0.001). These differences were more pronounced
255 during the laying stage (Pre-laying: unpigmented feathers, mean (95% CI) = 1.37 (0.14 –
256 2.6); pigmented feathers, mean (95% CI) = 0.04 (-0.04 – 0.11); Laying: unpigmented
257 feathers, mean (95% CI) = 3.44 (2.12 – 4.77); pigmented feathers, mean (95% CI) = 0.15 (-
258 0.03 – 0.33)) (F1,25 = 17.99, P < 0.001).
259 Finally, meshes with unpigmented feathers needed to be replenished more
260 frequently than those with pigmented feathers (unpigmented feathers: range: 0-4, mean ±
261 SE = 1.6 ± 0.5; pigmented feathers: range: 0-1, mean ± SE = 0.2 ± 0.1; F1,18 = 7.35, P =
262 0.014; Fig 1C). Thus, independently of the used variable, all results invariably suggest that
263 starlings preferentially chose unpigmented feathers, mainly during the laying stage.
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265 Green plant preferences
266 In the pre-laying stage, mass of plants in nests with and without plants offered did not differ
267 significantly (with experimental plants available: mean (95% CI) = 0.90 g (0.35 – 1.46);
268 without experimental plants available: mean (95% 5CI) = 0.44 g (0.15 – 1.74); F1,48 = 0.81,
269 P = 0.557). Moreover, nest stage did not affect mass of plants in starling nests (Pre-laying:
270 mean (%95CI) = 0.90 g (0.35 – 1.46); Laying: (95% CI) = 0.67 g (0.67 – 1.16)) (F1,26 =
271 0.32, P = 0.579), but laying date tended to explain a significant proportion of variance (F1,25
272 = 6.60, P = 0.051). Mass of plants found in nests during the laying stage increased as the
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273 season progressed, while the opposite trend was detected for the pre-laying stage (Fig 2A).
274 Percentage of vegetation coverage around nests did not significantly affect mass of plants
275 found in nests (mean (95% CI) = 78.93% (71.73 – 86.13); F1,22 = 2.77, P = 0.220).
276 We analyzed the starling preferences for experimental plants during the two nest
277 stages separately. In the pre-laying stage, prevalence of different experimental plant species
2 278 in starling nests did not differ (χ 4 = 3.13, P = 0.579; Fig 2B). However, starlings showed
2 279 some preferences during the laying stage (χ 4 = 14.79, P = 0.030; Fig 2B). L. amplexicaule
280 and M. vulgare were the experimental plants most frequently found in the nests, while A.
281 barrielieri and H. vulgare appeared with the lowest prevalence (paired comparisons: L.
2 2 282 amplexicaule – H. vulgare: χ 2 = 6.00, P = 0.014; M. vulgare – H. vulgare: χ 2 = 4.35, P =
283 0.037; Fig 2B). Similarly, the number of aromatic plant stems collected by starlings from
284 the containers were larger than that of non-aromatic plants (F4,435 = 4.73, P = 0.001; Fig 2C;
285 post-hoc LSD: L. amplexicaule – H. vulgare: P = 0.0001; M. vulgare – H. vulgare: P =
286 0.0002; A. clavatus – H. vulgare: P = 0.004; A. barrelieri – H. vulgare: P = 0.006; Fig 2C).
287 Thus, it is during the laying stage when preference for aromatic plants comes out.
288 Finally, antimicrobial activity assays of green plants offered to starlings showed that
289 A. barrelieri was the plant with the highest antimicrobial activity, followed by A. clavatus,
290 and L. ampleuxicaule. The lowest antimicrobial activity was shown by M. vulgare and H.
291 vulgare (Table 1).
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293 Discussion
294 The experimental results showed that starlings preferred unpigmented over pigmented
295 feathers and aromatic plants over non-aromatic plants for building their nests. Moreover,
296 these preferences were more clearly detected during the laying stage, when the hypothetical
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297 protective function of these nest materials for developing offspring would be more
298 important.
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300 Feather preferences
301 Feathers were more abundant in nests of starlings during the laying stage than during the
302 pre-laying stage. This is in accordance with the common yet often overlooked assumption
303 that nest building in birds does not end with the onset of laying, but continue during
304 posterior stages, likely to accommodate successive selection pressures (Hansell 2000).
305 Independently of the nesting stage, unpigmented feathers were more abundant than
306 pigmented feathers in starling nests. The main studied function of nest feathers concerns
307 their thermoregulatory properties (Møller 1984; Windsor et al. 2013), which for instance
308 would help to reduce clutch and brood cooling rates (Hilton et al. 2004). It is known that
309 pigmentation partially determines thermal insulation properties of structures in nature
310 (Hochscheid et al. 2002; Hetem et al. 2009; for a similar argument in avian eggs see Lahti
311 and Ardia 2016). However, previous studies dealing with insulation properties of feathers
312 in avian nests have not considered the potential effect of feather pigmentation (Hilton et al.
313 2004; Dawson et al. 2011; Windsor et al. 2013). This possibility is out of scope of the
314 present study. However, any hypothetical effect of feather pigmentation on nest
315 thermoregulation will mainly apply to open nests since external radiation reaching nest
316 contents inside holes is limited (Kilner 1999; Hunt et al. 2003; Avilés et al. 2008) and, thus,
317 would hardly apply to starling nests.
318 A second functional explanation for the preference of unpigmented feathers is
319 related to scenarios of sexual selection (Veiga and Polo 2005), with feathers in nests acting
320 as a courtship display affecting mate choice, and/or as a post-mating sexual signal eliciting
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321 differential reproductive investment in mates (Sanz and García-Navas 2011; but see Veiga
322 and Polo (2011)). Spotless starling females carry feathers to the nest in response to green
323 plants carried by males (Polo and Veiga 2006). We also found that feathers were more
324 abundant during the laying stage than during the pre-laying stage. This would be in
325 accordance with a role for feathers as sexual signals used by females to elicit differential
326 male investment because this period is closer to the early nestling stage, when contribution
327 to parental care by males is much needed (Soler et al. 2008). Moreover, amount of feathers
328 in starling nests is related to female experience (Polo and Veiga 2006), and the
329 experimental addition of feathers resulted in reduced nestling mortality (Veiga and Polo
330 2011). Thus, it is likely that feathers induced differential paternal investment in
331 reproduction (Veiga and Polo 2011).
332 Even if feather color preference is driven by sexual selection, it may also function in
333 additional scenarios. In this sense, trying to figure out possible scenarios explaining the
334 detected positive effects of experimental feathers reducing nestling mortality, Veiga and
335 Polo (2011) discussed the role of feathers as a possible antiparasitic material that would
336 reduce nestling mortality. Thus, it is even possible that the reason underlying the preference
337 for unpigmented feathers is their higher antimicrobial properties that have been
338 demonstrated in laboratory (Peralta-Sánchez et al. 2014) and in the field in nests of barn
339 swallows (Peralta-Sánchez et al. 2010). An alternative explanation for color composition of
340 nest-lining feathers is that it could mirror the availability or detectability of feathers of
341 different color in the nest surroundings. However, this possibility would not explain feather
342 color composition of nests in our study because pigmented and unpigmented feathers were
343 available ad libitum and easily locatable.
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344 Even knowing the starling preference for unpigmented feathers, and the ad libitum
345 availability of our experimental approach, very few nests (7 out of 48 nests) harbored only
346 unpigmented feathers, which suggest that starlings tried to incorporate feathers to the nest
347 following a certain combination of colors that they prefer. Interestingly, the detected
348 combination of feather colors changed, with a relative increase of unpigmented feathers as
349 the season progressed. Previous studies found that bacterial colonies from pigmented
350 feathers also have antibacterial activity (Peralta-Sánchez et al. 2014) and that the
351 abundance of pigmented feathers has a positive effect on phenotypic quality of spotless
352 starling nestlings (Ruiz-Castellano et al. unpublished). Moreover, antimicrobial activity of
353 bacterial colonies isolated from unpigmented feathers in nests of barn swallows was higher
354 in nests where experimental pigmented feathers were added (Peralta-Sánchez et al. 2014).
355 Thus, it is possible that certain combination of pigmented and unpigmented feathers in
356 nests of birds maximizes feather-derived antimicrobial activity. Moreover, environmental
357 conditions at the end of the breeding season favor bacterial growth in avian nests (Soler et
358 al. 2015; Møller et al. 2015). Therefore, it is possible that starlings tried to compensate such
359 increase in probability of bacterial infection by carrying to the nest feathers with higher
360 antimicrobial activity as the season progressed (Peralta-Sánchez et al. 2014). Further
361 research on antimicrobial properties of bacteria isolated from unpigmented and pigmented
362 feathers in nests of birds with different combinations of feather color is necessary to
363 explore this possibility.
364
365 Green plant preferences
366 Starlings also showed a preference for specific green plants to build their nests. Availability
367 of experimental plants did not affect plant abundance in their nests, but starlings that breed
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368 later used more plants than early breeders did. This positive association between laying date
369 and amount of green plants in the nest has also been found for European starlings (Clark
370 and Mason 1985; Gwinner and Berger 2005; Dubiec et al. 2013). It could be argued that
371 this is just the outcome of a higher accumulation of plants in nests that commenced egg
372 laying later. However, this explanation is unlikely for several reasons. First, because it is
373 possible that starlings, as some other bird species do, remove part of the old dried plants
374 after some days in the nest (Petit et al. 2002), and second, because for a given starling nest,
375 the amount of plants found during the laying stage was smaller than during the pre-laying
376 stage. In addition, it is known that female starlings remove from the nest the green plants
377 carried by males (Veiga and Polo 2012), which reflect male quality (Veiga et al. 2006).
378 This female behavior has been explained as a mean to difficult the assessment of male and
379 nest quality by neighboring rival females (Veiga and Polo 2012), which usually prospect
380 other nests to gather public information (Parejo et al. 2008). Removing green plants would
381 therefore prevent nest usurpation and reduce nest attractiveness for conspecific brood
382 parasites in European and spotless starlings (Sandell and Diemer 1999; Veiga and Polo
383 2012). Thus, it is possible that the relatively smaller amount of plants detected in starling
384 nests during the laying stage was the consequence of this female behavior. Another
385 explanation could be that males do not incorporate green material to the nest during the
386 laying stage, as has been claimed for European starlings (Gwinner 1997), and for some
387 other populations of spotless starlings (Veiga et al. 2006). However, this explanation is
388 unlikely since spotless starling males in our population continue carrying green material to
389 the nests during the laying and incubation stages (Ruiz-Castellano et al. 2016).
390 In agreement with a possible antimicrobial function of plants, we also found that
391 starlings did not show a marked preference for any of the offered plant species to be carried
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392 to their nests during the pre-laying stage, but during the egg-laying, males preferentially
393 selected aromatic plants. It is possible that, before egg laying males attract females to their
394 nests by carrying fresh green material with less emphasis on antimicrobial and antiparasitic
395 properties of plants (Hansell 2000). However, once reproduction has started, selecting
396 aromatic plants would have the additional advantage of protecting the nests against
397 parasites (Clark and Mason 1985) and microorganisms (Mennerat et al. 2009a). We know
398 that European starling males discriminate plant volatiles (Clark and Mason 1997) and that
399 this ability is maximized during the courtship and nest building stage (Clark and Smeraski
400 1990), when they select plant materials based on volatile compounds. Thus, it is possible
401 that spotless starling males also select plants with antimicrobial properties relying on their
402 volatile compounds. Most of the aromatic plants that we provided to starlings have strong
403 antimicrobial activity (see Table 1). The most preferred plant species appeared to be L.
404 amplexicaule, and not A. barrelieri that showed the highest antimicrobial activity. We
405 estimated antimicrobial activity of these plants by testing inhibitory potential of growth of
406 11 bacterial strains that include possible avian pathogens. Thus, although antimicrobial
407 activity against different bacterial strains usually covaries (Table 1; see also Al-Bakri and
408 Afifi 2007; Khalil et al. 2009), we cannot rule out the possibility that the most preferred
409 plants were active against some particular bacterial pathogens more abundant in the study
410 area. Additional analyses, ideally with bacteria isolated from nests, would be needed to
411 determine antimicrobial activity of nest plants against bacteria present in starling nests.
412 Independently of the reasons determining preferences for specific aromatic plants, the
413 detected higher amount of green plants during the pre-laying stage, as well as the
414 preferences for aromatic plants during the laying stage, suggest a primary role of this
18
415 material in sexual signaling and nest protection against pathogens, which may be more
416 prominent during the egg laying stage (see Rubalcaba et al. 2016).
417
418 Conclusion
419 Irrespective of differential functionality of the diverse nest materials, and of the possible
420 optimization of nest material composition, we have shown that starlings make a selection of
421 nest building materials in a possible adaptive way. We have shown this preference
422 regarding two common nest materials profusely employed by many bird species from
423 different orders and in different habitats to build their nests, i.e., green plants and feathers
424 (Hansell 2000; Dubiec et al. 2013). Furthermore, these two materials play pivotal roles in
425 the study of nest building behaviors, and may aid a better comprehension of self-medication
426 tool use by animals (Hansell and Ruxton 2008; Healy et al. 2008; De Roode et al. 2013).
427 Thus, our study help to better understand the adaptive functionality of nest building
428 processes and their involved behaviors.
429
430 Acknowledges
431 This work was supported by Spanish Ministerio de Ciencia e Innovación, European funds
432 (FEDER) (CGL2010-19233-C03-01, CGL2010-19233-C03-03, CGL2013-48193-C3-1-P,
433 CGL2013- 48193-C3-3-P). MRR received a postdoc from the program “JAE-Doc” and
434 CRC had a predoctoral grant from the Spanish Government. GT was supported by Ramón y
435 Cajal program (Spain).
436
437 Data Accessibility
19
438 Analyses reported in this article can be reproduced using the data provided by Ruiz-
439 Castellano C, Tomás G, Ruiz-Rodríguez M, and Soler JJ. (2017) Data from: Nest material
440 preferences by spotless starlings. Behavioral Ecology. In Dryad Digital Repository,
441 http://dx.doi.org/10.5061/dryad.q6ng9
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28
592 Table 1: Antimicrobial activity of the different green plants offered to spotless starlings for
593 nest building. We show the scores for halo transparency, halo size, number of bacteria for
594 which tested plants demonstrated antimicrobial activity, and the index scores for each
595 tested plant.
Number Halo Halo of Index Plant Bacteria transparency size inhibited score bacteria A. barrelieri B. licheniformis 2 4 5 14.990 S. aureus 2 4 L. inocua 2 4 L. monocytogenes 2 1 E. faecalis 2 1 A. clavatus B. licheniformis 1 4 2 -0.054 S. aureus 1 4 L. inocua 0 0 L. monocytogenes 0 0 E. faecalis 0 0 L. ampleuxicaule B. licheniformis 1 4 1 -4.198 S. aureus 0 0 L. inocua 0 0 L. monocytogenes 0 0 E. faecalis 0 0 M. vulgare B. licheniformis 1 2 1 -5.369 S. aureus 0 0 L. inocua 0 0 L. monocytogenes 0 0 E. faecalis 0 0 H. vulgare B. licheniformis 1 2 1 -5.369 S. aureus 0 0 L. inocua 0 0 L. monocytogenes 0 0 E. faecalis 0 0 596
29
597 Fig 1: (A) Feather color composition (± CI 95%) in spotless starling nests during the pre-
598 laying stage, with or without experimental feathers available. (B) Number of pigmented and
599 unpigmented feathers (± CI 95%) in spotless starling nests during the pre-laying and the
600 egg laying stages. (C) Mean number of times (± CI 95%) that meshes containing the
601 experimental pigmented and unpigmented feathers offered to spotless starlings needed to be
602 replenished.
603
604 Fig 2: (A) Relationships between laying date and log10 of plant mass (g) of spotless
605 starling nests recorded both at the pre-laying and at the egg laying stages. Regression lines
606 are shown. (B) Prevalence of different plant species (aromatic: L. amplexicaule, M.
607 vulgare, A. clavatus, A. barrelieri; non-aromatic: H. vulgare) in spotless starling nests at
608 the pre-laying and egg laying stages. (C) Number of experimental plant fragments (± CI
609 95%) of the different species collected from containers by spotless starlings during the
610 three weeks that plants were available.
611
612
30
614 Fig 1:
615
616
617
31
618 Fig. 2
619
620
621
622
32