TY - JOUR
AU - Włodarczyk,, Radosław
AB - Abstract Eggshell pigmentation is generated by 2 major pigments, biliverdin and protoporphyrin. The latter is mostly deposited in red, brown, and black egg spots and it has been hypothesized that greater expression of egg spottiness (as measured by the number, area, and coloration of spots) may act as an honest signal of female quality for males (sexual signaling hypothesis, SSH). The important assumption of the SSH is that eggshell pigmentation correlates with phenotypic and genetic components of female quality, although phenotypic quality of females may also be under environmental control. The aim of this study was to test for the associations of protoporphyrin-based egg pigmentation with both phenotypic and genetic female traits and environmental variables (microhabitat and urbanization) in a common rallid species, the Eurasian Coot (Fulica atra). We found that the total number of egg spots was positively associated with female condition (size-corrected body mass) and expression of a putative bare-part ornament (frontal shield). The same measure of spottiness negatively correlated with the level of physiological stress in females. No evidence was found for associations between egg spottiness and genetic traits in females (neutral heterozygosity and polymorphism of pathogen recognition receptors, the Major Histocompatibility Complex), but there was a linear increase in the expression of egg spottiness over the breeding season, which may suggest that it is regulated by food availability. Our study indicates that protoporphyrin-based pigmentation of eggs reflects female phenotypic traits (condition, stress, and ornament expression) in the Eurasian Coot, although it remains to be established whether it plays any signaling role and whether it is driven by sexual selection in this species. Resumen La pigmentación de la cáscara del huevo es generada por dos pigmentos principales, biliverdina y protoporfirina. El último es mayormente depositado en manchas de huevo rojas, marrones y negras y se ha hipotetizado que una mayor expresión de las manchas del huevo (medida como el número, el área y la coloración de las manchas) puede actuar como una señal honesta de la calidad de la hembra para los machos (hipótesis de la señalización sexual, HSS). El supuesto importante de la HSS es que la pigmentación de la cáscara del huevo se correlaciona con componentes fenotípicos y genéticos de la calidad de la hembra, aunque la calidad fenotípica de la hembra puede también estar bajo control ambiental. El objetivo de este estudio fue evaluar las asociaciones de la pigmentación por protoporfirina del huevo tanto con rasgos fenotípicos y genéticos de la hembra como con variables ambientales (micro-hábitat y urbanización) en la especie de rálido común, Fulica atra. Encontramos que el número total de manchas del huevo estuvo positivamente asociado con la condición de la hembra (masa corporal corregida por tamaño) y con la expresión de un ornamento putativo de la parte desnuda (escudo frontal). La misma medida de las manchas se correlacionó negativamente con el nivel de estrés fisiológico en las hembras. No encontramos evidencia de asociaciones entre las manchas del huevo y los rasgos genéticos en las hembras (heterocigosis neutral y polimorfismo de receptores de reconocimiento de patógenos, el Complejo Mayor de Histocompatibilidad), pero hubo un aumento lineal en la expresión de las manchas del huevo a lo largo de la estación reproductiva, lo que puede sugerir que está regulada por la disponibilidad de alimento. Nuestro estudio indica que la pigmentación por protoporfirina de los huevos refleja los rasgos fenotípicos de la hembra (condición, estrés y expresión de los ornamentos) en Fulica atra, aunque aún debe establecerse si juega algún rol de señalización y si está condicionada por selección sexual en esta especie. Lay Summary • There is equivocal support for direct associations between maternal quality and deposition of protoporphyrin pigmentation (dark spots and blotches) in avian eggshells. • Research on protoporphyrin eggshell pigmentation has primarily focused on a single avian order (Passeriformes). • We examined associations of protoporphyrin-based eggshell pigmentation with female phenotypic and genetic traits in a non-passerine species, the Eurasian Coot. • Deposition of protoporphyrin in eggshells (total number per area of egg spots) positively correlated with female condition and expression of a putative bare-part ornament (frontal shield), while it was negatively associated with the level of physiological stress. • Protoporphyrin-based eggshell pigmentation acts as a reliable signal of female phenotypic (but not genetic) traits in the Eurasian Coot. • Signaling properties of protoporphyrin-based egg coloration are likely to largely differ between different evolutionary lineages of birds. INTRODUCTION There is a general agreement that coloration patterns of avian eggs have evolved under varying selective pressures in different phylogenetic lineages of birds (Cherry and Gosler 2010). In many species, eggs have a cryptic coloration that enhances predation avoidance, and predatory pressure has long been recognized as one of the most important evolutionary determinants of eggshell pigmentation (Kilner 2006). Higher predation rates are generally thought to select for darker eggs (with darker background and greater spottiness, although it may depend on the egg-laying substrate), but eggs of many species of birds (mainly ground-nesting birds that nest in sites with no vegetation cover) also face high solar radiation pressures, so they have a tradeoff between camouflage and overheating (Gómez et al. 2016). Eggshell pigmentation has also been hypothesized to serve many other adaptive functions, many of which are not necessarily mutually exclusive. First, specific coloration patterns may help identification of an individual’s own eggs in species exposed to intra- or inter-specific brood parasitism (Davies and Brooke 1989). Egg coloration may also regulate light transmission through the eggshell, filtering harmful ultraviolet radiation on the one hand (Gómez et al. 2018), and allowing greater light transmission to enhance embryo development under low light exposure on the other (Maurer et al. 2015). Finally, eggshell pigmentation may play an antimicrobial activity role (Ishikawa et al. 2010), regulate eggshell permeability (Higham and Gosler 2006), or improve structural properties of eggshells and compensate for reduced eggshell thickness (Gosler et al. 2005). Although basic between-species differences in egg coloration can be likely explained with ancient diversification in breeding ecology or nest characteristics (Kilner 2006) and appear to be regulated by female-specific genes (Gosler et al. 2000), there is increasing evidence for the environmental regulation in the expression of avian eggshell pigmentation. For example, long-term variation in eggshell coloration of Eurasian Reed Warbler (Acrocephalus scirpaceus) covaried with spring weather conditions, such as temperature and rainfall (Avilés et al. 2007). Because deposition of pigments in the eggshell is primarily dependent on the ability to acquire them from diet or physiological capacity to synthesize them by females, these associations were suggested to reflect the direct effect of weather on female physiological status (e.g., via elevated physiological stress due to inclement weather) or indirect effects on female condition mediated through weather-related food availability (Avilés et al. 2007). There is also strong evidence for direct associations between maternal quality and eggshell pigmentation (Moreno et al. 2006, Siefferman et al. 2006, Hanley et al. 2008, Soler et al. 2008), which constitutes an important assumption for the sexual signaling hypothesis (SSH) (Moreno and Osorno 2003). In general, the SSH assumes that males may adjust their contribution to offspring care accordingly to the female quality, as perceived from the expression of egg pigmentation (Moreno and Osorno 2003). Although this hypothesis is based on several important predictions (e.g., males must be able to perceive variation in eggshell phenotype, they must adjust their behavior accordingly, and this adjustment must be adaptive), the basic assumption of the SSH is that eggshell pigmentation correlates with phenotypic and genetic components of female quality, rather than with environmental factors (although phenotypic quality of females may also be under environmental control). So far, most support for the SSH comes from research that focused on biliverdin, which is one of the 2 major eggshell pigments. Biliverdin generates blue or green background coloration of avian eggshells and chemically it has strong antioxidant properties (Jansen and Daiber 2012). Thus, it has been hypothesized that deposition of biliverdin in eggs may be achieved at a certain cost and females, which allocate more biliverdin pigments in eggs, are likely to impair their capacity to control free radicals (Moreno and Osorno 2003, Morales et al. 2008), although adaptive significance of this mechanism is still under debate (Reynolds et al. 2009, Cherry and Gosler 2010). In contrast, the second major eggshell pigment, protoporphyrin, is a pro-oxidant that acts as a natural metabolite intermediate in the biosynthesis of heme. Consequently, extensive deposition of protoporphyrin in eggshell may indicate high capacity of a female to sustain elevated levels of pro-oxidants and, thus, signal high tolerance to oxidative stress (Moreno and Osorno 2003). Protoporphyrin is primarily deposited in the dark (usually red, brown, or black) spots or blotches within avian eggshells, but studies on the associations between maternal quality and protoporphyrin eggshell pigmentation have, so far, provided mixed results (Martínez-de la Puente et al. 2007, de Hierro and de Neve 2010, Stoddard et al. 2012, Hargitai et al. 2016a, 2016b). Nevertheless, past research has primarily focused on the taxa from a single avian order (passerines) and much broader phylogenetic sampling (including non-passerine avian orders) is needed to draw robust conclusions on the adaptive significance of protoporphyrin-based egg pigmentation. The major aim of this study was to test for the associations of female phenotypic and genetic traits with protoporphyrin-based egg pigmentation in a common rallid (non-passerine) species, the Eurasian Coot (Fulica atra). Coots lay whitish eggs with conspicuous black spots that are putatively produced by the protoporphyrin pigments (Figure 1). Following the key assumption of the SSH, we hypothesized that stronger expression of egg spottiness (in terms of total number, total area, and reflectance of egg spots) should indicate better phenotypic and genetic quality of females (but not necessarily of males). To test for this hypothesis, we examined associations of egg spottiness with female condition (size-adjusted body mass and blood hemoglobin concentration), physiological stress (heterophil to lymphocyte ratio), putative ornament expression (size of the frontal shield), neutral heterozygosity, and polymorphism of pathogen recognition receptor genes (the number of alleles across duplicated Major Histocompatibility Complex [MHC] loci). We expected females in better condition and under lower physiological stress to lay more pigmented eggs because they should have greater resources available to sustain elevated levels of pro-oxidants and, thus, should be able to produce more protoporphyrin pigments. We also expected more pigmented eggs in females with greater expression of a putative ornament, as ornament expression should reflect better condition and, possibly, higher capacity to tolerate oxidative stress (e.g., Henschen et al. 2016). Finally, we expected greater egg pigmentation in females with higher polymorphism at the key immune receptors and heterozygosity of neutral loci, as better protection against pathogens and higher genome-wide heterozygosity are associated with a broad spectrum of fitness-related traits in birds (e.g., Forstmeier et al. 2012, Dunn et al. 2013) and, thus, may also correlate with oxidative stress resistance. To examine environmental components in egg pigmentation patterns of coots we also tested for the spatial (microhabitat of nesting sites and urbanization level) and temporal (intra-seasonal) adjustments in egg spottiness. FIGURE 1. Open in new tabDownload slide Variation in the spottiness pattern of Eurasian Coot eggs. Eggs with 306 (A) vs. 667 (B) spots and 2.26 cm2 (A) vs. 8.17 cm2 (B) total spot area are shown. FIGURE 1. Open in new tabDownload slide Variation in the spottiness pattern of Eurasian Coot eggs. Eggs with 306 (A) vs. 667 (B) spots and 2.26 cm2 (A) vs. 8.17 cm2 (B) total spot area are shown. METHODS Study Site and General Field Procedures The study was conducted in central Poland in 2017–2018. Breeding populations of Eurasian Coot were monitored at 3 sites that differed in the level of urbanization. The first study site was located in the central part of a large city, Łódź (51.77°N, 19.47°E; 293 km2 total area, ~700,000 inhabitants). The area was characterized by compact settlement and strong anthropogenic pressure, and breeding habitat for coots was highly transformed (low reed cover). Eurasian Coots from this urban population were previously found to show a spectrum of behaviors generally characteristic for urban faunas (so-called “urban wildlife syndrome”; Evans et al. 2010), including elevated boldness and aggression, better resistance to external stressors, and better adaptation to the exploitation of human-derived resources (Minias 2015b, Minias et al. 2018a). The second study site was located at the peripheries of Łódź city, where Eurasian Coots bred in semi-natural habitats (moderate reed cover) with moderate to low anthropogenic pressure (suburban population). Finally, the third study site consisted of 2 nearby fishpond complexes in Sarnów (51.85°N, 19.10°E) and Żeromin (51.62°N, 19.59°E) (rural population). Fishponds were located on private properties with restricted access, resulting in low anthropogenic pressure. Breeding habitat was natural-like and characterized by shallow water and extensive areas of reed cover. The study was based on a randomly selected sample of 100 coot nests (29–38 nests per population). All the nests were found during egg laying or shortly after clutch completion. Laying date was assigned to 5-day periods, starting from March 15 to June 15. For each nesting site we collected the following information: (1) predominant vegetation type within 1 m of the nest assigned to 6 categories: I—Phragmites reeds, II—Typha angustifolia reeds, III—Typha latifolia reeds, IV—low emergent vegetation (Carex, Acorus, Scirpus), V—woody plants (mainly Salix), VI—no vegetation cover; (2) distance from nest to open water (for nests located away from vegetation on open water, the distance was set to zero); (3) distance from nest to shore (for nests located at the water–land border, the distance was set to zero); and (4) water depth at the nest (±0.1 m). In total, we captured 77 adult coots. Most birds (n = 66) were captured during incubation on nests either with noose traps made of monofilament nylon line or by hand (exclusively in the urban population). This sample was used to assess condition and putative bare-part ornament expression. The remaining 11 birds were captured during the nonbreeding period preceding egg spottiness measurements and, thus, were only used for genetic analyses to avoid any bias resulting from seasonal variation in condition and ornament expression. Upon capture, wing length was measured with a stopped ruler (±1 mm) and body mass was measured with an electronic balance (±1 g). All birds were marked with a metal ring and a plastic neckband. Eight marked individuals were associated with 2 different nesting attempts included in our sample, either within the same season or in different seasons. Sex of all birds was determined with molecular methods, according to the protocol described elsewhere (Minias et al. 2018a). Our sample was represented by 35 females and 42 males (13–32 individuals per population). Egg Spottiness Pattern To assess egg volume and spottiness we randomly selected 5 eggs per clutch, although in 8 nests only 3–4 eggs were available due to early egg losses, which resulted in a final sample size of 489 eggs. First, each egg was cleaned with a cloth and all vegetation particles removed from the egg’s surface. Then, we photographed each egg following recommendations by Gómez and Liñán-Cembrano (2017); each photograph was analyzed with SpotEgg software (Gómez and Liñán-Cembrano 2017), a free tool that runs over MATLAB (MathWorks, Natick, Massachusetts, USA). Eggs were placed above a gray standard (Lastolite Ezybalance, 30 cm, 18% reflectance) and photographs (5,202 × 3,464 pixels) were taken from a distance of ~30 cm above the eggs in RAW format using a Canon EOS 6D digital camera. A standard color chart (ColorChecker Passport, X-Rite, Grand Rapids, Michigan, USA) was also included in the scene of each photograph to normalize and linearize the image. As conventional digital cameras employ the so-called radiometric transfer function (a nonlinear function) to produce visually appealing images at the expense of a reduction of the contrast in low-contrast areas, images needed to be linearized with respect to irradiance to obtain meaningful reflectance results. After linearizing the images with SpotEgg, they were mapped into equivalent reflectance images by using the known information from the pixels in the reflectance target. Then, to characterize properly (in a standardized way) the spottiness of each egg and to calculate egg volume, we selected a scale that was included in the color chart and also, and more importantly, selected the 4 parameters (radius average filter = 2.010513; spot detector sensitivity = 0.1967802; minimum spot size = 0.025 mm2; background fill threshold = 0.15311) to be applied to the entire dataset. This parameterized algorithm employed by SpotEgg basically relies on defining optimized spatially variant thresholds to segment spots from background (more details in Gómez and Liñán-Cembrano 2017) and it allows the analysis of all images using the same parameters, thus, increasing repeatability when compared with other subjective methods. The background constancy option was set to zero. Each egg was characterized using 4 variables (egg volume, total number of egg spots, total area of egg spots, and average egg spot reflectance) that were extracted from SpotEgg software and used in the analyses. Measurements of Phenotypic Traits To quantify phenotypic traits in coots we used 2 condition measures and the level of putative bare-part ornament expression. As a first measure of condition we used size-corrected body mass that was calculated as the scaled mass index (SMI) using the formula developed by Peig and Green (2009): SMI=Mi[L0Li]bSMA where Mi and Li are the body mass and the linear body measurement of individual i, respectively; bSMA is the scaling exponent estimated by the standardized major axis regression of body mass on linear body measurement; L0 is the mean value of the linear body measurement for the study population. Among the 3 collected morphological measurements (wing length, total head length, and tarsus length), wing length showed the strongest log-log correlation with body mass (r = 0.80, P < 0.001, n = 69), indicating that it best explained fraction of mass associated with structural size (Peig and Green 2009). We computed bSMA for both sexes jointly using 100,000 bootstraps in RMA software (Bohonak 2004). The mean value of wing length L0 = 215.66 mm was used to estimate SMI. Whole-blood hemoglobin concentration was used as the second measure of condition in coots. Hemoglobin concentration is a general measure of blood oxygen-carrying capacity in vertebrates and it has been shown to correlate with a wide spectrum of fitness-related traits, as well as to depend negatively on the level of ectoparasitism and positively on diet quality in birds (reviewed in Minias 2015c). Although hemoglobin concentrations may change substantially (usually decrease) in laying females due to several physiological processes associated with egg production (Wagner et al. 2008a, 2008b), no female was captured at the laying stage within our dataset. We measured hemoglobin concentration spectrophotometrically with a portable HemoCue Hb 201 + photometer (HemoCue, Ängeholm, Sweden) using ~5 μL of blood collected from a tarsal vein into a disposable HemoCue microcuvette. Physiological stress was quantified for a subsample of 58 individuals as a relative ratio of heterophils to lymphocytes (hereafter “H/L ratio”). In a stressful environment, the number of lymphocytes in the peripheral blood decreases, while the number of heterophils increases, as they are released from bone marrow (Johnstone et al. 2012). This process of leucocyte trafficking is mediated through stress hormones and, thus, H/L ratio is commonly used as a simple, but relatively robust measure of physiological stress in vertebrates (Davis et al. 2008), despite the fact that it can be affected by a wide variety of external and internal factors, such as pollution, inclement weather events, habitat fragmentation, urbanization, increased breeding effort, and social stressors (Minias et al. 2018b), which may hamper accurate interpretations of its variation. For the purpose of the measurements, a drop of blood was collected from the ulnar vein and transferred to a slide, where a smear of one cell layer was made. Blood sampling was conducted within half an hour of capture to avoid any effect of acute handling stress on H/L ratio measurements (Davis 2005). Each smear was air-dried, stained using the May–Grünewald–Giemsa method, and scanned at 1,000× magnification under a light microscope. A random sample of 100 leucocytes from each smear was classified into 5 cell types: heterophils, lymphocytes, eosinophils, basophils, and monocytes. The H/L ratio was calculated by dividing the number of heterophils by the number of lymphocytes. To reduce variability, all blood smears were assessed by one of the authors (RW) and the measurements showed high repeatability (R = 0.85, P < 0.001), as assessed with the intra-class correlation coefficient calculated for repeated measurements of 20 randomly chosen smears. Since the distribution of the H/L ratio was strongly right-skewed (skewness: 1.26), it was log-transformed prior to analyses, which brought the distribution reasonably close to normal (skewness: 0.08). Finally, we quantified expression level of a putative bare-part (non-plumage) ornament, the frontal shield. Eurasian Coots are monochromatic and sexually monomorphic in plumage, but both sexes develop a conspicuous and sexually dimorphic (larger in males; Minias 2015a) white fleshy frontal shield that extends from the bill onto the head crown. In general, the size of the frontal shields in rallids expands prior to the breeding season and these changes were found to be testosterone-dependent in both sexes (Eens et al. 2000). Although we are aware of no experimental data on the signaling role of frontal shield in Eurasian Coots, shield size has been shown to reliably signal dominance, social status, fighting ability, and condition in several other rallids (Crowley and Magrath 2004, Alvarez et al. 2005, Dey et al. 2014). The width of the frontal shield was measured with calipers to the nearest 0.1 mm in all captured coots and used as a proxy of ornament expression. Measurements of Genetic Traits We measured diversity at both neutral (microsatellite) and adaptive (MHC) genetic markers in coots. Neutral heterozygosity and MHC polymorphism belong to well-established measures of genetic quality in birds and they were shown to correlate with fitness-related traits and sexually selected ornament expression in a wide spectrum of avian taxa (e.g., von Schantz et al. 1996, García-Navas et al. 2009, Forstmeier et al. 2012, Dunn et al. 2013), although we have no direct evidence that these traits predict fitness in our study populations of Eurasian Coot. Neutral heterozygosity was assessed across 10 microsatellite loci that were either originally developed for other rallid species (Tasmanian Nativehen [Gallinula mortierii], Buchan 2000; King Rail [Rallus elegans], Brackett et al. 2013; and California Black Rail [Laterallus jamaicensis], Molecular Ecology Resources Primer Development Consortium et al. 2009) or found to be highly conservative across a wide range of non-passerine bird species (Dawson et al. 2010). Nuclear DNA was extracted from blood samples using Bio-Trace DNA Purification Kit (EURx, Gdańsk, Poland) and PCR amplifications were conducted according to the original protocols. An ABI/Hitachi 3500 (Applied Biosystems, Foster City, California, USA) sequencer was used for the fragment size analysis and the size of alleles was determined with GeneScan TM 600 LIZ Standard (Applied Biosystems) in Geneious 10.0.5 (Biomatters, Auckland, New Zealand) software. We found 4–44 alleles per microsatellite locus across all 3 populations (n = 77 individuals) and observed heterozygosity of these loci ranged from 0.16 to 0.92. Deviations from Hardy–Weinberg equilibrium (HWE) were assessed for each locus in each population with the exact tests (Guo and Thompson 1992) that were run with a Markov chain method (chain length: 1,000,000; dememorization: 100,000) in Arlequin 1.3.5.2 (Excoffier and Lischer 2010). One locus deviated significantly (after Bonferroni correction) from the HWE, but this deviation was recorded only in a single population (urban) and, thus, the locus was not excluded from the analyses. We found no evidence for pairwise linkage disequilibrium between loci, as assessed with Genepop 4.1.2 (Rousset 2008) and the frequency of null alleles at each locus was low (<0.05), as estimated with Cervus 3.0.3 (Kalinowski et al. 2007). Heterozygosity was calculated across all 10 loci for all individuals. To assess polymorphism at the MHC we used Fuat-Ex2Fw and Fuat-Ex2Rv primers developed by Alcaide et al. (2014) to genotype MHC class II B exon 2 (gene fragment that forms a peptide-binding groove of MHC molecules) in the Eurasian Coot. PCR amplifications were conducted in a final volume of 20 μL containing 10 μL of 2X HotStarTaq Plus Master Mix Kit (Qiagen, Venlo, Netherlands), 50–150 ng of genomic DNA, and 0.2 μM of each primer; the protocol followed Alcaide et al. (2014). Amplifications were completed using fusion primers containing Illumina adapter sequences, a 7 base pair (bp) barcode that indicated sample identity, and a pair of original MHC primers. All PCR products were purified and their concentrations were determined with Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Equimolar quantities of PCR products were pooled to form a library using NEBNext DNA Library Prep Master Mix Set for Illumina (New England Biolabs, Ipswich, Massachusetts, USA). The library was sequenced using Illumina v2 Kit at a 2 × 250 bp paired-end Illumina MiSeq platform. Processing of Illumina MiSeq data was conducted using Amplicon Sequencing Analysis Tools (AmpliSAT) web server developed by Sebastian et al. (2016). Pair-ended reads were merged with AmpliMERGE, while de-multiplexing, clustering, and filtering of Illumina reads was conducted with AmpliSAS (Sebastian et al. 2016). Default parameters for Illumina data were used for clustering (1% substitution errors, 0.001% indel errors, and 25% minimum dominant frequency) and filtering (discarding chimeras and sequences that had less than 3% frequency) steps. The final results yielded 100% reproducibility of alleles between technical replicates (n = 10 individuals). MHC alleles were aligned in Geneious 10.0.5 and intron regions were removed from the alignments, retaining a full length (270 bp) of MHC class II exon 2. The number of alleles recorded per individual ranged from 1 to 6, consistent with the number of 3 duplicated MHC class II B loci as previously found by Alcaide et al. (2014) in the Eurasian Coot. Since we could not calculate the exact heterozygosity across all 3 MHC loci, we used the number of alleles recorded per individual as a measure of MHC diversity. Statistical Analyses Egg volume and egg spottiness parameters (total number of egg spots, total area of egg spots, and average egg spot reflectance) were entered as response variables in separate general linear mixed models (GLMM). First, we ran GLMMs testing associations of egg volume/spottiness with nesting site characteristics, which were entered as fixed factors (type of emergent vegetation) or covariates (distance to open water, distance to shore, and water depth). Laying date was also entered as a covariate, population was entered as a fixed factor, and nest identity and year were entered as random factors in these models. In the analyses of the total number and total area of egg spots we also included egg volume as an additional covariate, because of significant correlations between these traits (see Results for details). Second, we ran GLMMs to test for associations of egg volume/spottiness with phenotypic traits (frontal shield size, SMI, and blood hemoglobin concentration), physiological stress (log-transformed H/L ratio), and genetic traits (MHC diversity and microsatellite heterozygosity) of birds. Each explanatory variable (except for both genetic traits) was analyzed using a separate model, as most of them differed in sample sizes (e.g., data from some individuals were used only for the analyses of genetic, but not phenotypic, traits or stress). Similar to the nesting site choice models, we entered laying date as a covariate and population as a fixed factor, while nest identity and year were entered as random factors. Wing length was entered as an additional covariate to control for structural size of birds, while individual identity was entered as an additional random factor since 8 individuals were associated with 2 nesting attempts within our dataset. All models were run separately for males and females. GLMMs were run using the lme4 package (Bates et al. 2015) developed for the R statistical environment (R Development Core Team 2013). We used the car package (Fox and Weisberg 2011) to obtain Wald χ 2 (W) statistics and to infer statistical significance (P values) for all predictors. To obtain more parsimonious best-fitted models, we used the dredge function from the MuMIn R package (Bartoń 2020). The models were selected based on the Akaike’s information criterion corrected for small sample size (AICc). For each analysis we presented the results of full, best-fitted, and null model. Pairwise intercorrelations between different egg parameters were also tested with GLMMs to avoid pseudoreplication. Within-nest repeatability of egg parameters was calculated as the intra-class correlation coefficient using the irr R package (Gamer et al. 2012). Differences between coefficients of variation were tested using the z-score test in Real Statistics (Zaiontz 2012–2019) Excel (Microsoft, Redmond, Washington, USA) add-in. All values are reported as means ± standard error (SE). RESULTS Egg Volume and Egg Spottiness Pattern Total number and total area of egg spots showed significantly higher between-egg variation than egg volume and average egg spot reflectance (all P < 0.001), as assessed with the coefficients of variation (Table 1). Egg volume and total area of egg spots showed highest within-nest repeatability (R = 0.70 ± 0.04 in both cases). Within-nest repeatability of the total number of egg spots and average egg spot reflectance was lower (R = 0.61 ± 0.04 and R = 0.58 ± 0.05, respectively), but all repeatability estimates were highly significant (all P < 0.001). Egg volume correlated positively with the total number of spots (β = 0.011 ± 0.001; W = 75.87, df = 1, P < 0.001) and total area of spots (β = 0.57 ± 0.18; W = 10.12, df = 1, P = 0.001), but not with the average spot reflectance (W = 2.10, df = 1, P = 0.15). Total area of egg spots was positively associated with total number of spots (β = 0.0054 ± 0.0002; W = 604.2, df = 1, P < 0.001) and negatively associated with average egg spot reflectance (β = −5.30 ± 1.68; W = 9.95, df = 1, P = 0.002), but there was no significant association between the total number and average reflectance of egg spots (W = 2.77, df = 1, P = 0.10). TABLE 1. Descriptive statistics for egg volume and egg spottiness parameters in the Eurasian Coot. Trait . Mean . SE . Range (min–max) . Coefficient of variation (CV) . Egg volume (cm3) 37.03 0.19 26.86–57.46 11.55 Total number of egg spots 363.9 6.3 58–920 37.85 Total area of egg spots (cm2) 3.51 0.05 1.31–8.17 31.09 Average egg spot reflectance 0.138 0.001 0.08–0.27 19.11 Trait . Mean . SE . Range (min–max) . Coefficient of variation (CV) . Egg volume (cm3) 37.03 0.19 26.86–57.46 11.55 Total number of egg spots 363.9 6.3 58–920 37.85 Total area of egg spots (cm2) 3.51 0.05 1.31–8.17 31.09 Average egg spot reflectance 0.138 0.001 0.08–0.27 19.11 Open in new tab TABLE 1. Descriptive statistics for egg volume and egg spottiness parameters in the Eurasian Coot. Trait . Mean . SE . Range (min–max) . Coefficient of variation (CV) . Egg volume (cm3) 37.03 0.19 26.86–57.46 11.55 Total number of egg spots 363.9 6.3 58–920 37.85 Total area of egg spots (cm2) 3.51 0.05 1.31–8.17 31.09 Average egg spot reflectance 0.138 0.001 0.08–0.27 19.11 Trait . Mean . SE . Range (min–max) . Coefficient of variation (CV) . Egg volume (cm3) 37.03 0.19 26.86–57.46 11.55 Total number of egg spots 363.9 6.3 58–920 37.85 Total area of egg spots (cm2) 3.51 0.05 1.31–8.17 31.09 Average egg spot reflectance 0.138 0.001 0.08–0.27 19.11 Open in new tab Spatial and Seasonal Variation Egg volume differed significantly between populations (W = 7.24, df = 2, P = 0.027; Supplemental Material Table S1), as urban coots laid smaller eggs (35.58 ± 0.55 cm3) than birds from suburban (37.82 ± 0.65 cm3) and rural (38.11 ± 0.60 cm3) populations, although these pairwise differences were marginally nonsignificant (P = 0.067 and P = 0.068, respectively). The difference in egg volume between suburban and rural populations was highly nonsignificant (P = 0.999). Egg volume was also found to vary with the type of emergent vegetation (W = 12.41, df = 5, P = 0.030; Supplemental Material Table S1), and this variation was primarily driven by significantly larger egg volumes in nests with no vegetation cover when compared with nests located in Typha latifolia (39.90 ± 0.60 cm3 vs. 34.06 ± 0.50 cm3; P = 0.013). Egg volume was not associated with laying date and other nesting site characteristics (Supplemental Material Table S1). After controlling for variation in egg volume, we found that total number and total area of egg spots increased with laying date, indicating that late breeders laid eggs with higher number of spots (β = 2.01 ± 0.59; W = 11.65, df = 1, P < 0.001) and larger area of spots (β = 0.025 ± 0.005; W = 22.38, df = 1, P < 0.001) (Figure 2, Supplemental Material Table S1). Average spot reflectance was not associated with laying date (Supplemental Material Table S1). None of egg spottiness parameters correlated with nesting site characteristics or differed between populations (Supplemental Material Table S1). FIGURE 2. Open in new tabDownload slide Seasonal variation in the total number (A) and total area (B) of egg spots (residuals against egg volume) in Eurasian Coots. Regression lines (solid lines) with 95% confidence intervals (dashed lines) are shown. FIGURE 2. Open in new tabDownload slide Seasonal variation in the total number (A) and total area (B) of egg spots (residuals against egg volume) in Eurasian Coots. Regression lines (solid lines) with 95% confidence intervals (dashed lines) are shown. Egg Spottiness vs. Phenotypic and Genetic Traits Total number of egg spots was positively associated with the size of the frontal shield in female coots (β = 17.46 ± 8.70; W = 4.02, df = 1, P = 0.045; Figure 3, Supplemental Material Table S2) and female SMI (β = 0.94 ± 0.41; W = 5.35, df = 1, P = 0.021; Figure 4A, Supplemental Material Table S3). There was also indication for a positive association between female SMI and total area of egg spots, as inferred from the full model (β = 0.007 ± 0.003; W = 5.23, df = 1, P = 0.022; Figure 4B), but this relationship was not supported by the best-fitted model (Supplemental Material Table S3). Total number of egg spots was negatively associated with female H/L ratios (β = −489.9 ± 207.7; W = 5.56, df = 1, P = 0.018; Figure 5A) and a similar, although marginally nonsignificant, association was found for total egg spot area (β = −2.28 ± 1.30; W = 3.07, df = 1, P = 0.080; Figure 5B, Supplemental Material Table S5), indicating that coots under stronger physiological stress laid less-spotted eggs. Neither total number or total area of egg spots correlated with blood hemoglobin concentration of females (Supplemental Material Table S4). Egg volume and average egg spot reflectance correlated with none of the phenotypic traits in females (Supplemental Material Tables S2–S5). No significant associations were found between egg volume or egg spottiness parameters and phenotypic traits of males (Supplemental Material Tables S2–S5). Also, we found no association between egg volume or egg spottiness parameters with genetic traits (MHC diversity and heterozygosity at neutral loci) of either male or female coots (Supplemental Material Table S6). FIGURE 3. Open in new tabDownload slide Relationship of the total number of egg spots (residuals against egg volume) with the size of frontal shield in female Eurasian Coots. Regression line (solid line) with 95% confidence interval (dashed lines) is shown. FIGURE 3. Open in new tabDownload slide Relationship of the total number of egg spots (residuals against egg volume) with the size of frontal shield in female Eurasian Coots. Regression line (solid line) with 95% confidence interval (dashed lines) is shown. FIGURE 4. Open in new tabDownload slide Relationships of the total number (A) and total area (B) of egg spots (residuals against egg volume) with scaled mass index in female Eurasian Coots. The relationship for the total area of egg spots was not retained in the best-fitted model (see Supplemental Material Table S3). Regression line (solid line) with 95% confidence interval (dashed lines) is shown. FIGURE 4. Open in new tabDownload slide Relationships of the total number (A) and total area (B) of egg spots (residuals against egg volume) with scaled mass index in female Eurasian Coots. The relationship for the total area of egg spots was not retained in the best-fitted model (see Supplemental Material Table S3). Regression line (solid line) with 95% confidence interval (dashed lines) is shown. FIGURE 5. Open in new tabDownload slide Relationships of the total number (A) and total area (B) of egg spots (residuals against egg volume) with heterophil/lymphocyte (H/L) ratio in female Eurasian Coots. The relationship for the total area of egg spots was marginally nonsignificant (see Supplemental Material Table S5). Regression lines (solid line) with 95% confidence intervals (dashed lines) are shown. FIGURE 5. Open in new tabDownload slide Relationships of the total number (A) and total area (B) of egg spots (residuals against egg volume) with heterophil/lymphocyte (H/L) ratio in female Eurasian Coots. The relationship for the total area of egg spots was marginally nonsignificant (see Supplemental Material Table S5). Regression lines (solid line) with 95% confidence intervals (dashed lines) are shown. DISCUSSION Our study provided a clear correlational support for associations between maternal phenotypic traits and egg patterning in the Eurasian Coot. We found that the total number of egg spots was positively associated with female condition (size-corrected body mass, SMI) and expression of a putative bare-part ornament (frontal shield). It also negatively correlated with the level of physiological stress (H/L ratios) in females. Weaker evidence for similar associations (positive with SMI and negative with H/L ratios) was found for the total area of egg spots. At the same time, we found no support for associations between egg spottiness and putative measures of female genetic quality. Finally, neither phenotypic nor genetic traits of males correlated with egg spottiness. Positive associations of egg spottiness with maternal condition, physiological stress, and ornamental expression provide a possible indication for a signaling role of protoporphyrin deposition in the eggshell. In general, a reliable condition-dependent signal may arise only if the expression of a trait carries a certain cost to an individual and if this cost is higher for low-quality than high-quality individuals (Zahavi 1975, Andersson and Iwasa 1996). A putative cost associated with production of protoporphyrin-based egg maculation stems from a pro-oxidative activity of this pigment and from the fact that high levels of protoporphyrin attained during egg production may cause oxidative damage to the females (Moreno and Osorno 2003). Thus, females with low tolerance to oxidative stress may not be able to efficiently cope with high organismal concentrations of protoporphyrin and, consequently, they should maintain low protoporphyrin levels and produce less-spotted eggs, although some other mechanisms could be also in operation, especially if eggshell protoporphyrin was synthesized de novo in the shell gland instead of being derived from blood (see Hargitai et al. 2017 for details). While evidence for a direct relationship between the strength of protoporphyrin-based egg pigmentation and female oxidative status is lacking, it has been recently found that female Great Tits (Parus major) with higher antioxidant capacity laid eggs with more homogeneous distribution of protoporphyrin-based pigmentation (Giordano et al. 2015), but other study on the same species showed that eggs with darker protoporphyrin-based spots contained lower concentrations of antioxidants in the yolk (Hargitai et al. 2016b). In general, research on Paridae failed to provide convincing support for positive associations between expression of egg pigmentation and female quality (Martínez-de la Puente et al. 2007, Stoddard et al. 2012, Hargitai et al. 2016b; but see Holveck et al. 2019). Research on other passerine species led to similar conclusions, as no significant (or significantly negative) associations between protoporphyrin-based egg pigmentation and female quality were usually reported (Walters and Getty 2010, Hargitai et al. 2016a). On the other hand, stronger protoporphyrin pigmentation correlated with higher hatching success in the Barn Swallow (Hirundo rustica), suggesting that it can predict fitness (Corti et al. 2018). Similar research is virtually lacking in non-passerine birds and, in general, our study on the Eurasian Coot is one of few that provided clear support for the positive associations between the expression of protoporphyrin-based egg pigmentation and female phenotypic traits. Although a positive association between egg spottiness and maternal phenotype in the Eurasian Coot suggests that protoporphyrin deposition in the eggshell may have an adaptive role, its significance is far from clear. One of the most debated hypotheses on the adaptive role of eggshell coloration predicts that males could possibly perceive maternal quality from the expression of egg pigmentation and adjust their contribution to offspring care accordingly (sexually selected eggshell coloration hypothesis; Moreno and Osorno 2003). Although the hypothesis initially gained some experimental support (Soler et al. 2008, Sanz and García-Navas 2009), most later studies in a variety of avian taxa failed to replicate the results and provided little evidence for adjustments in paternal investment in relation to eggshell coloration (Hanley and Doucet 2009, Walters and Getty 2010, Honza et al. 2011, Johnsen et al. 2011, Stoddard et al. 2012, Fronstin et al. 2016). It also remains an open question whether egg spottiness can have any general adaptive significance for the Eurasian Coot. Although coots nest in reed-type vegetation, which should provide some protection for clutches, the species is known to suffer heavy losses of eggs due to avian predators, mostly corvids and harriers (Zduniak 2006). Thus, stronger pigmentation of eggs could possibly provide more efficient camouflage, but contrary to this hypothesis we found no variation in egg spottiness patterns between microhabitat characteristics at the nesting sites and between urban and rural landscapes, which differ markedly in the available habitats. Another adaptive value of protoporphyrin-based pigmentation, except for crypsis, may be an enhancement of eggshell strength (structural-function hypothesis; Gosler et al. 2005). It has been shown in Great Tits that more protoporphyrin is deposited in thinner parts of eggshells and the darkness of pigment indicated the degree of thinning (Gosler et al. 2005). Under assumptions of this hypothesis, coots of higher phenotypic quality could invest more in the production and deposition of protoporphyrin in eggshells to improve their durability, but we failed to collect any data that could serve to test this hypothesis. Finally, it has been suggested that the risk of overheating may be an important selective agent in the evolution of egg pigmentation (Gómez et al 2018). However, we find this mechanism unlikely to drive variation in egg spottiness in our study populations, first because sun radiation is not extreme in Central Europe, and second because we found that more-spotted eggs were laid at the end of the season when sun radiation is higher, being contrary to the predictions of this hypothesis. It has been recently suggested that expression of protoporphyrin-based egg pigmentation can also be affected by food availability (Hargitai et al. 2013, 2018). Although we did not measure food availability in our coot study populations, a clear increase in the spottiness of coot eggs during the breeding season may be likely associated with changes in the quantity and quality of available food resources. Coots from our population are short-distance migrants and in spring they arrive at breeding grounds as soon as ice cover disappears from the territories, usually in early or mid-March. The diet of coots mostly consists of macrophytes and aquatic macroinvertebrates (Snow and Perrins 1998), availability of which may be limited at the beginning of the breeding season. In general, macrophytes from the European shallow lakes start to grow rapidly in April and their biomass reaches a peak no sooner than late May (Villa et al. 2018). Similarly, seasonal dynamics of macroinvertebrate communities often parallel the development of submerged vegetation (van den Berg et al. 1997). For example, a study of coot diet in the Netherlands showed a peak in the biomass of water-surface insects in late May and early June, and their availability was a key determinant of breeding success (Brinkhof 1997). Consequently, coots that start laying at the end of April or beginning of May (peak laying period) are expected to be in better nutritional condition than early breeders. Assuming that nutritional condition could affect oxidative tolerance of coots, as was found in a wide spectrum of other avian species (Costantini et al. 2007, Giordano et al. 2015, Ruuskanen et al. 2017), a seasonal increase in egg spottiness that we observed in our study population could be mediated by foot availability. On the other hand, a few experimental studies on birds have demonstrated a negative relationship between food supplementation and protoporphyrin concentration in eggshells (Duval et al. 2016, Hargitai et al. 2018) and, thus, ecological and physiological mechanisms responsible for seasonal increase in egg spottiness of coots need further exploration. Despite this, our study on the Eurasian Coot seems to support the hypothesis that protoporphyrin-based egg pigmentation may be primarily determined by a combination of maternal traits and environmental factors. Although we found positive associations of egg pigmentation with multiple phenotypic traits (condition, ornamental expression, and physiological stress) of female coots, our study provided no support for associations between eggshell spottiness and female genetic traits. Specifically, we found that egg pigmentation was not related to neutral heterozygosity and polymorphism of MHC genes, which determine the spectrum of antigens (and consequently pathogens) against which an acquired immune response can be triggered. At the same time, we acknowledge that heterozygosity–fitness correlations (HFCs) in natural vertebrate populations are often weak (Chapman et al. 2009) and may require much more extensive sampling data than we were able to acquire. However, consistent with our results, most studies have failed to convincingly demonstrate a link between egg coloration and female genetic quality (reviewed in Riehl 2011). We also found no significant association of egg pigmentation with phenotypic or genetic traits of male coots, which is congruent with female sex-linked inheritance of avian eggshell patterning (Gosler et al. 2000). So far, covariation between egg pigmentation and male quality has been rarely reported and usually attributed to differential allocation of resources by females paired with males of different quality, parallel effects of environmental variables (e.g., territory quality) on both parents, or assortative mating (Sanz and García-Navas 2009, Badás et al 2017). In conclusion, we showed that protoporphyrin-based pigmentation acts as a reliable signal of female phenotypic traits in the Eurasian Coot. As far as we are aware, this is one of the few studies that provided clear support for the positive associations of protoporphyrin-based egg pigmentation with maternal condition and (putative) ornament expression in birds. At the same time, we found seasonal variation in egg spottiness, suggesting that protoporphyrin-based egg coloration may primarily be determined by a combination of maternal traits and environmental factors. In general, our study reinforces the view that signaling properties of protoporphyrin-based egg coloration are likely to largely differ between different evolutionary lineages of birds. ACKNOWLEDGMENTS We want to thank Ewa Pikus for help with microsatellite and MHC genotyping. We thank 4 anonymous reviewers and the Associate Editor for constructive comments on the earlier drafts of the manuscript. Funding statement: No funding was received for this work Ethics approval and consent to participate: The study was conducted in accordance with the current laws of Poland, where it was performed under the permission of the Local Bioethical Commission for Animal Welfare in Łódź and General Environmental Protection Directorate in Łódź. Author contributions: PM designed the study. PM and RW collected field samples. RW analyzed leucocyte profiles. JG analyzed egg spottiness patterns. PM performed statistical modeling. All authors wrote the manuscript and revised it for intellectual content. 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TI - Egg spottiness reflects female condition, physiological stress, and ornament expression in a common rallid species
JO - The Auk: Ornithological Advances
DO - 10.1093/auk/ukaa054
DA - 2020-12-24
UR - https://www.deepdyve.com/lp/oxford-university-press/egg-spottiness-reflects-female-condition-physiological-stress-and-CtrkPx050M
VL - 137
IS - 4
DP - DeepDyve
ER -