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Paper wasps are darker at high elevation
Journal Pre-proof Paper wasps are darker at high elevation André R. de Souza, Angie Z. Mayorquin, Carlos E. Sarmiento PII:
S0306-4565(19)30626-6
DOI:
https://doi.org/10.1016/j.jtherbio.2020.102535
Reference:
TB 102535
To appear in:
Journal of Thermal Biology
Received Date: 9 November 2019 Revised Date:
21 January 2020
Accepted Date: 9 February 2020
Please cite this article as: de Souza, André.R., Mayorquin, A.Z., Sarmiento, C.E., Paper wasps are darker at high elevation, Journal of Thermal Biology (2020), doi: https://doi.org/10.1016/ j.jtherbio.2020.102535. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Author statement The authors declare that they have no conflicts of interest associated with the contents of this manuscript. Specimens were collected following Colombian National Park regulations. Regarding CRediT roles: André de Souza: conceptualization, Methodology, Investigation, Data curation, Writing - Original Draft, review and editing. Angie Z Mayorquin: Investigation, Data curation, Methodology, Formal analysis, Investigation, Writing Original Draft, review and editing. Carlos E Sarmiento: conceptualization, Formal analysis, Resources, Writing - Original Draft, review and editing.
Paper wasps are darker at high elevation
André R. de Souzaa, Angie Z. Mayorquinb and Carlos E. Sarmientoc *
a
Departamento de Biologia, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Av. Bandeirantes 3900, 14040-901 São Paulo, Brazil. e-mail: [email protected]
b
Universidad Nacional de Colombia, Instituto de Ciencias Naturales, Cr 30 No. 45 03
edif 425, of. 303, Bogotá, Colombia. e-mail: [email protected]
c
Universidad Nacional de Colombia, Instituto de Ciencias Naturales, Cr 30 No. 45 03
edif 425, of. 303, Bogotá, Colombia. e-mail: [email protected]
* Corresponding author
Abstract: High mountains are harsh environments in which colder temperatures and higher levels of UV-B radiation are common. These abiotic conditions strongly affect animals` biology, often constraining their survival and reproduction. As a result, adaptations to live in such habitats are expected to evolve. Body color is thought to be adaptive to the environment that animals experience. Tegument melanization improves heat gain and provides photoprotection. Therefore, at high elevation, ectotherms are expected to be darker (well-melanized). We test this prediction in the paper wasp Agelaia pallipes (Hymenoptera: Vespidae), a species distributed across an elevational gradient in the Colombian Andes. We used Malaise traps and sampled a total of 146 wasps along nine elevations, ranging from 2,600-3,380 m above sea level. Standard digital photography was used to measure the body luminance and colour patterning in different body parts of dry-preserved specimens. There was striking variation in body luminance (darker and lighter), color patterning (patched, smoothed, homogeneous) and surface texture (shiny and matte), but the kind and degree of variation depended on the body part examined. Wasps from higher elevations had darker thoraces, confirming our prediction. Besides, at high elevation, the frequency of wasps with a matte rather than a shiny face strongly increased. Overall, our findings support the thermal melanism hypothesis and suggest that intraspecific color variation might be an adaptation to the environment of paper wasps.
Keywords: High mountain; intraspecific colour variation; thermal melanism; UV-B radiation; thermoregulation.
1. Introduction
High mountain habitats exhibit extreme abiotic conditions and animals living in such places often differ phenotypically from lowland equivalents (Hodkinson, 2005; Keller et al., 2013). The steepness of these elevational gradients is particularly relevant for analyzing the adaptiveness of intraspecific variation in species that occur from low to high elevations (Körner, 2007). Body color is thought to be adaptive to the environment that animals experience (Burtt, 1981; Cloudsley-Thompson, 1999), so that the presence of multiple colour morphs within a species is expected to broaden the range of environmental conditions under which individuals are able to survive and reproduce (Forsman et al., 2008; Caesar et al., 2010). Across animal taxa, changes in body colour according to the elevation have been described and the ultimate causes of such phenotypic clines debated (Beetles: Mikhailov, 2001; Vipers: Castella et al., 2013; Lizards: Reguera et al., 2014; Ants: Bishop et al., 2016; Law et al., 2019; Grasshoppers: Köhler et al., 2017; Crickets: Kuyucu et al. 2018). At high elevation, the most influential abiotic variables on animals` biology are the lower temperatures and the higher levels of solar radiation (Körner, 2007). In insects, cold constrains the activity levels, as these ectotherms depend mostly on extrinsic factors for thermorregulation. Additionally, UV-B light, a kind of ionizing radiation comprising a small component of the solar electromagnetic spectrum, is strongly absorbed by nucleic acids (Ravanat et al., 2001) and a number of deleterious effects have been reported for insects (Beard, 1972; Bothwell et al., 1994; McCloudx). To deal with these harsh abiotic
conditions, insects may show adaptive color variation. Eumelanin is a black pigment widespread across animal taxa and other phyla (Riley, 1997; Sugumaran, 2002; Pinkert and Zeuss, 2018). Insects at high elevation tend to have well-melanized and therefore dark (low luminance) bodies (Mikhailov, 2001; Bishop et al., 2016; Köhler et al., 2017; Kuyucu et al., 2018). Darkening the body surface improves the passive thermorregulation ability (following the thermal melanism hypothesis, Watt, 1968; Clusella-Trullas et al., 2007) and provides protection against UV-B radiation (The photo-protection hypothesis, Law et al., 2019), therefore suggesting the adaptive meaning of insect melanism at high elevation. Body melanism is supposed to be adaptive in environments other than high elevations, as it improves resistance to desiccation in drier environments (following the melanism-desiccation hypothesis, Kalmus, 1941) and provides camouflage, resistance to parasites and pathogens and protection against UV-B radiation in warmer and more humid environments (following the Gloger`s rule, Delhey, 2019). Adaptive body melanism can be detected even at a microclimatic gradient. For example, examining ant assemblages along the vertical strata in a tropical forest showed that darker ants are more common in the canopy, as these are more likely to deal with the relatively higher levels of desiccation and UV-B radiation in the canopy (Law et al., 2019). Thus, body melanism may provide a solution for multiple selective pressures. In Paper wasps (Hymenoptera: Vespidae), body colour variation is known to mediate social communication processes such as quality signaling and individual identity signaling (Cervo et al., 2015). Relatively less investigated is the possibility that wasp body colour evolved as an adaptation to environmental factors such as temperature or UV-B radiation. De Souza et al. (2017a) examined a set of dry-preserved museum species of Polistes paper wasps from different regions of the world and
established and association between thorax luminance and annual mean temperature of collection region, leading the authors to conclude that darker paper wasps have selective advantage in colder environments. Badejo et al. (2018) examined a set of ethanol-preserved Vespula vulgaris paper wasps from different climatic areas and found that higher latitudes (colder environments) may select for darker paper wasps. Both investigations (De Souza et al., 2017a; Badejo et al., 2018) support the thermal melanism hypothesis. On the other hand, body coloration of Vespa velutina paper wasps, examined by visual assessment, seems not to be related to climatic conditions (Perrard et al., 2014). To our knowledge, whether or not body coloration tracks the elevation of wasp distribution has not been tested. The swarm-founding paper wasp Agelaia pallipes have a Neotropical distribution, from Costa Rica to northern Argentina (Richards, 1978). In addition to this wide latitudinal occurrence, these wasps are often distributed from lowlands up to high mountain forests more than 3,000 m above sea level, such as in the Colombian Andes (Rodríguez-Jimenez and Sarmiento, 2008). According to Sarmiento (1993), their colonies are populous, holding up to 16,500 adult individuals. Nests are ovoid, of about 50 cm of maximum diameter, and many combs are surrounded by an envelope composed by several irregular layers. The nests can be either exposed to direct solar radiation or sheltered within trunk holes. During the day, the wasps often stay over the exposed nest surface or nearby the nest entrance, potentially receiving direct solar radiation. Rodríguez-Jimenez and Sarmiento (2008) provided anecdotal observations suggesting that these wasps seem to be darker at high elevation, but no formal assessment was performed.
Here, we investigate whether paper wasp coloration is selected by the environment at which wasps are exposed. If so, we predict that wasp coloration is associated with elevation. First, we describe intraspecific variation in the colour of different body parts in female A. pallipes. Then, we test if this phenotypic variation tracks the elevation of population distribution. From these results, we made inferences about the adaptive meaning of body colour in relation to the main abiotic selective forces at high elevation, the colder temperatures and the higher levels of UV-B light.
2. Material and Methods
2.1. Study place and wasp collection The study was performed in the Santuario Nacional de Flora y Fauna de Iguaque (5º42’21" N, 73º27’25" W, coordinates of the fourth altitude sampling site 2,850 m, close to the visitor´s center of the park). This national park is located in the eastern mountain range of the Colombian Andes, with elevation ranging between 2,400 and 3,800 m above sea level. Climate data at the site are not available but there is a strong negative relationship between elevation and temperature (R2= 0.935) and changes along the year are negligible (12.2°C - 13.6°C at 2690m) (IDEAM, 2017). The Colombian western mountain range has average temperature between 17.3°C for 2000m (12.321.0°C) and 11.3°C (5.9-15.1°C) for 3000m. The vegetation comprises oak forest, cloud forest, and paramo. Wasps were sampled by performing a set of field expeditions between March of 2000 and October of 2001. In each expedition, four malaise traps (collecting jars filled with 95% ethanol) spaced by 100 m from each other were set out for around 10 days,
after then the wasps were collected from the traps. These sampling expeditions were performed at eight different elevations, described in Table 1. Thus, the elevation of collection sites ranged between 2,600 and 3,380 m above sea level and the distances from each other between ranged between 400m and 3.2 km. Between 11 and 20 female wasps were sampled in each elevation, performing a final sample of 146 individuals across the gradient. Each wasp was removed from the ethanol, individually pinned and then set to dry eight days at room temperature (around 21°C). After then, image acquisition was performed.
2.2. Image acquisition We used standard digital photography protocols (reviewed in Stevens et al., 2007). Basically, each wasp was examined by a researcher who was blind to the collection place of each individual. This researcher captured images of four different body parts: frontal view of the clypeus, dorsal view of the mesoescutum, dorsal medial view of the second abdominal tergite and ventral medial view of the second abdominal sternite (Fig. 1 and Fig. 2). In this way, we obtained 8-bit RAW digital images by using a Leica Q2 camera adapted to a Leica S8APO stereoscope which includes a ring Leica LED illumination system at constant intensity. The stereoscope and the camera settings were kept constant (stereoscope setting: augment 4 times; camera settings: ISO = 100, f= 6,3, time of aperture = 1/160 seconds). Wasps were set at a distance of around 2 cm from the stereoscope objective. The white balance was calibrated with a 18% grey card (Kodak) and the same card was placed in the frame of each specimen as a control. We used LIGHTROOM, version 4.1 (Adobe Systems Inc.) to colourcorrect each image and them we exported them to a TIFF format.
2.3. Measuring body coloration Each picture was transferred to the Image J software, version 1.51j8 (Rasband, 2018) in order to measure the body luminance. This was performed in the RGB colour space. In this system, the luminance value of a given pixel ranges between 0 and 255, in which 0 indicates black (the darkest value) while 255 indicates white (the brightest value). The luminance of each body part was calculated as the average of eight randomly selected pixels. The mesoescutum of A. pallipes often have patterns of yellowish stripes over the black background (Fig. 2). These yellowish stripes are darker or lighter, larger or smaller, and sometimes absent, according to the wasp examined. To account for this striking variation, we performed a new set of measurements in the mesoescutum images in which we estimated the luminance of the yellowish area only, calculated as the average of four randomly selected pixels in this area. We also estimated the relative area of the mesoescutum occupied by the yellowish stripes. Of note, some wasps, especially those from higher elevations lack yellow stripes on the mesoescutum (as predicted by the Thermal melanism Hypothesis, see results). These wasps whose mesoescutum was entirely black were excluded from the analysis of the luminance of the yellowish area of the mesoescutum (31 out of 149 wasps were excluded in these analyzes). Finally, we also categorized by visual assessment wasp clypeus as shinning (Fig. 2, left) or matte (Fig. 2, right). To estimate the error associated with our luminance measurement protocol, a set of 10 wasps had the body parts measured independently by two researchers. Then, the repeated measurements were compared according to Sennar (1999). In this way, this error was 1% for both the mesoescutum and the yellow stripes, 2% for the clypeus and
5% for both the tergite and the sternite. These values are considered low (Sennar, 1999), so our measurements are highly repeatable.
2.4. Statistical analyses Descriptive statistics regarding variation in the color traits is reported as mean value, standard deviation and coefficient of variation (= standard deviation/mean). The inferential statistics was performed in different ways, as follow. To test whether there are differences in the luminance values between the different body parts (clypeus, mesoescutum, tergite and sternite) we applied the nonparametric Kruskal-Wallis test, as the assumptions required for parametric analyses were not met (even after several transformation trials). To identify which body parts are more or less luminant than the others, we applied post-hoc tests using Siegel and Castellan´s (1988) approach. To test whether luminance values in a given body part covaries with luminance values of the other body parts we applied the nonparametric Spearman`s rank correlation test. To test if the luminance of the different body parts is associated with elevation, we applied linear regression analyses. The required assumptions and outlier detection for these regressions (global fitness, skewness, kurtosis, link function, and heteroscedasticity) were previously tested using Peña and Slate`s (2006) approach based on Neyman smooth test. As a result, only the luminance values of the mesoescutum needed to be log-transformed to meet the assumptions of the regression analyses. Outliers are defined here as atypical data and not as error, these were identified also using the gvlma package approach of deletion statistics and observed in the graphic options of projections such as normal Q-Q plot, residuals vs fitted plot, half-normal Cook’s distance plot, and the
standardized residuals vs the fitted values plot (Peña and Slate, 2006; 2019); outliers were progressively removed until regression assumptions were met; outliers were identified in all variables, no more than five individuals were removed from an analysis and in no case these were clustered from a single altitude. All analyses were conducted using the software R 3.6.1. (R Core Team, 2019), using the packages Pgirmess (Giraudoux et al., 2018) and gvlma (Peña and Slate 2019). The level of significance was set at 5%. The RAW data used in this study is available as Electronic supplementary material ESM1.
3. Results
We found interindividual differences in body colour of A. pallipes (Fig. 2). Variation in body luminance was more evident for the mesoescutum (coefficient of variation, CV = 19%), followed by tergite (CV = 17%), sternite (coefficient of variation = 16%), and the clypeus (CV = 14%). The clypeus always looked homogeneously black, but there was variation in its surface texture, as some wasps had a shiny while others a matte clypeus. On the other hand, the mesoescutum may be entirely black or may had a patched coloration, as up to four stripes (0, 2 or 4) of yellowish coloration with a variable size (mean area of yellowish stripes relative to mesoescutum total area = 7%, standard deviation = 4%, CV = 67%), and luminance (Mean luminance = 95, standard deviation = 19, CV = 20%) could be present over the black background. A heterogeneous coloration was also noticed in the tergite and sternite, as both areas had a more or less smoothed pattern of yellowish and brownish areas. Thus, interindividual variation in body coloration is evident for the body surface texture (clypeus), luminance
values (clypeus, mesoescutum, tergite, sternite) and color patterning (mesoescutum, tergite and sternite). We also found differences in the luminance of the different body parts (Kruskall-Wallis test: H3 = 358.06, p = 0.001, Fig. 3A). Specifically, the darkest body part was the clypeus (Mean luminance = 53), followed by the mesoescutum (Mean luminance = 60), tergite (Mean luminance = 71) and the sternite (Mean luminance = 95). Thus, wasps are darker at the anterior (clypeus, mesoescutum) rather than the posterior (tergite, sternite) portion of the body, and they are darker at the dorsal (mesoescutum, tergite) rather than the ventral (sternite) portion of the body. Overall, the luminance between the different body parts was not correlated. The only exception was the weak positive association between the luminance of the mesoescutum and the tergite (Spearman`s rank correlation: clypeus vs. mesoescutum: r = -0.102, N = 145, p = 0.221; clypeus vs. tergite: r = -0.032, N = 142, p = 0.703; clypeus vs. sternite: r = 0.023, N = 144, p = 0.789; mesoescutum vs. tergite: r = 0.180, N = 143, p = 0.031; mesoescutum vs. sternite: r = 0.072, N = 145, p = 0.390; tergite vs. sternite: r = 0.111, N = 143 , p = 0.185). We found evidence for an association between body luminance and elevation of wasp collection, but such relationship depended on the wasp body part examined (Fig. 3B-G). Thus, the elevation of wasp collection was negatively associated with mesoescutum luminance (r2 = 0.18, b= -1.2e-04, F1,144 = 32.81, p = 0.001, Fig. 3C), but no association was detected between the elevation of wasp collection and the clypeus luminance (r2 = 0.01, b= 0.002, F1,143 = 1.08, p = 0.299, Fig. 3B), tergite luminance (r2 = 0.01, b= 0.005, F1,141 =
2.27, p = 0.134, Fig. 3D) or the sternite luminance (r2 = 0.01, b= 0.008, F1,143 = 3.51, p = 0.063, Fig. 3E). In other words, at higher elevations, wasp mesoescutum is darker, but the luminance of wasp clypeus, tergite and sternite seems not to be related to the elevation. Of note, even though the luminance in the clypeus did not track the elevation, the proportion of individuals with a matte rather than shiny clypeus dramatically increased at high elevations (Spearman`s rank correlation: r= 0.7319, N = 8, p = 0.0254, Fig. 3H). We also found that the elevation of wasp collection was negatively associated with the luminance of the yellowish area of the mesoescutum (r2 = 0.09, b= -0.02, F1,113 = 12.96, p = 0.001, Fig. 3F). Besides, as the elevation of wasp collection increases, the relative proportion of the yellowish pigmentation in the mesoescutum decreases (r2 = 0.23, b= -7,3e-5, F1,139 = 43.8, p = 0.001, Fig. 3G). Thus, at the higher elevations, the yellowish area of the mesoescutum is smaller and darker. Of note, all wasps from Lagunillas, the highest collection site, had entirely black mesoescutum as no yellow stripes were present.
4. Discussion
Agelaia pallipes paper wasps have a high degree of intraspecific variation in body colour, both at the interindividual (between individuals) and intraindividual (between different body parts) levels. Some of this variation is associated with the elevation. The thorax, specifically the mesoescutum, of A. pallipes paper wasps is darker at higher elevations. This is likely the result of changes in the colour of the yellowish
stripes over its surface as they are darker and smaller at high elevations. Darkening the body surface is often due to the deposition of melanin (Sugumaran, 2002) and this might be interpreted as an adaptation to deal with the colder temperatures at harsh environments (Valverde and Schielzeth, 2015). The thermal melanism hypothesis (Watt, 1968; Clusella-Trullas et al., 2007) proposes that darker morphs are better at converting solar radiation in heat than their brighter equivalents. Thus, darker ectothermic animals have an advantage in colder environments, because they can heat faster and become active before others. Darker morphs of several insects indeed increase in frequency at high elevations (Majerus, 1998; True, 2003; Clusella-Trullas et al., 2007). Besides, there are records in insects that the melanization related to environmental temperature often occurs in dorsal body parts that are well-exposed to direct solar radiation and are nearby the flight muscles, such as the wings of butterflies (Watt, 1968; Guppy, 1986) and the mesoescutum of other paper wasps (De Souza et al., 2017a). UV-B radiation may provoke a number of deleterious effects in insects (Beard, 1972; Bothwell et al., 1994; McCloud and Berenbaum, 1999). Thus, becoming darker at higher elevations might also be interpreted as an adaptation to deal with the increased levels solar radiation at these harsh environments, because melanin deposition on the cuticle provides photoprotection (True, 2003). The facial and abdominal coloration, although variable, was not associated with the elevation. Thus, not all variation in wasp body color tracks environmental conditions, as it has been reported for some other wasps (Perrard et al., 2014; De Souza et al., 2017a). A number of other factors might account
for this variation, such as interspecific signaling (Vidal-Cordeiro et al., 2012), intraspecific signaling (Cervo et al., 2015) or even neutral variation (De Souza et al., 2017b). The A. pallipes paper wasps exhibited darker dorsal surface and a lighter ventral surface. This countershading is a widespread pattern across animal taxa (Cuthill et al., 2016). It might have evolved due to a number of reasons, such as improvement of the passive thermoregulation ability, protection against UV-B radiation and self-shadow concealment (Caro, 2005). Thus, providing additional ways by which wasp body color might potentially be an adaptive trait. Researches often try to identify a single key function to animal external appearance (Caro, 2017), but animal coloration may experience multiple selection pressures (Lindstedt et al., 2009) in specific body parts (De Souza et al., 2017a). This might be the case A. pallipes paper wasps, as the relationship between the body coloration and the elevation of wasp collection depends on the body part examined. Thus, despite body coloration of insects and other animals is generally studied by sampling one or a few body parts, we suggest that more detailed information (getting data from many different body parts, for example, Perrard et al., 2014; Williams, 2007) might be desirable if we are to better understand the evolution of animal colour. Variation in the facial (clypeal) surface texture in A. pallipes was evident, as we could clearly assign each wasp as having a shiny or a matte face. Besides, wasps tended to be matte-faced at high elevations. All else being equal, matte rather than shiny surfaces are presumably expected to be less luminant (because shiny surfaces are more reflexive). Thus, the increase in the proportion of wasps with a matte face at high elevations could potentially improve passive thermoregulation. However, in our study, the luminance in the clypeus did not track the elevation of wasp collection even though
the frequency of matte-faced wasps improved up to 100% at the highest elevation. One possibility is that our measurement protocol was not accurate enough to capture the subtle effect the body surface texture may have had in the luminance of already well-melanized body parts. Variation in the paper wasps body coloration is complex, ranging from uniform to smoothed and patched distribution. This body coloration is often considered to be a result of cuticular pigmentation (melanin, pterin), but our findings of matte dark and shiny dark surfaces highlights for the possibility of structural coloration in paper wasps (Chavarría-Pizarro et al., 2010), this is when colour is produced not from pigments, but from the differential light reflection of the ultra‐ or microstructure surface of the cuticle (Shawkey and D`Alba, 2017).
5. Conclusion
Our study shows that the coloration of A. pallipes paper wasp varies with elevation. Concretely, thorax dorsal coloration is darker in higher elevation, presumably to improve heating of muscles associated with flight. By contrast, coloration of other parts such as head and abdomen did not vary with elevation, except for clypeus which becomes more matte with elevation. These findings suggest that wasps are darker at high elevations in order to improve their thermoregulation and protect them from UV-B radiation
6. Acknowledgements Authors thanks members of the Laboratorio de Biología Comparada de Insectos of the Universidad Nacional de Colombia for their support during data gathering. This
work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (grant number 2015/05302-0 to AR de Souza) and by the National University of Colombia Hermes project 8367 (support to CES and AZM).
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Figure Legends
Table 1. Details of field expeditions to collect A. pallipes paper wasps in different elevations. Fig. 1. Sketch of wasp body indicating parts in which colour variation was examined. Fig. 2. Examples of body colour variation in A. pallipes paper wasps from different elevations. Pictures illustrate some variation in the female clypeus (although consistently black, it ranges from matte to shiny as the elevation increases, comprising differences in surface texture), mesoescutum (note the patched coloration and the variation in the number, size, and luminance of the yellowish stripes, so that they are all reduced as the elevation increases), tergite and sternite (note the variation of brownish and yellowish smoothed patterns). Fig. 3. Body colour variation in A. pallipes and its relation with the elevation of collection. In A, within-box lines shows the median, the whiskers show the minimum and maximum values and different letters indicate statistical differences between the
classes. From B to G, each black dot refers to a single individual and the lines represent a significant association.
Electronic supplementary material ESM1. RAW data used in this study.
Collection site Cabaña Chaina El Nispero Site three La Planada and Cabaña Carrizal Site five Cerro Pan de Azúcar Quebrada Carrizal Lagunillas
Elevation (meters above sea level) 2.600 2.730 2.795 2.850 2.975 3.300 3.350 3.380
Time of collection May/2001 Oct/2001 Mar/2000 Jan/2001 and Oct/2001 Mar/2000 Mar/2001 Jan/2001 Mar/2001
• High mountains exhibit harsh abiotic conditions, like low temperatures and higher levels of UV-B radiation • Tegument melanization (darkening) improves heat gain and provides photoprotection • Females of Agelaia pallipes are darker at high elevations • Intraspecific colour variation might be an adaptation to the environment where of paper wasps live.
S0306-4565(19)30626-6
DOI:
https://doi.org/10.1016/j.jtherbio.2020.102535
Reference:
TB 102535
To appear in:
Journal of Thermal Biology
Received Date: 9 November 2019 Revised Date:
21 January 2020
Accepted Date: 9 February 2020
Please cite this article as: de Souza, André.R., Mayorquin, A.Z., Sarmiento, C.E., Paper wasps are darker at high elevation, Journal of Thermal Biology (2020), doi: https://doi.org/10.1016/ j.jtherbio.2020.102535. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Author statement The authors declare that they have no conflicts of interest associated with the contents of this manuscript. Specimens were collected following Colombian National Park regulations. Regarding CRediT roles: André de Souza: conceptualization, Methodology, Investigation, Data curation, Writing - Original Draft, review and editing. Angie Z Mayorquin: Investigation, Data curation, Methodology, Formal analysis, Investigation, Writing Original Draft, review and editing. Carlos E Sarmiento: conceptualization, Formal analysis, Resources, Writing - Original Draft, review and editing.
Paper wasps are darker at high elevation
André R. de Souzaa, Angie Z. Mayorquinb and Carlos E. Sarmientoc *
a
Departamento de Biologia, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Av. Bandeirantes 3900, 14040-901 São Paulo, Brazil. e-mail: [email protected]
b
Universidad Nacional de Colombia, Instituto de Ciencias Naturales, Cr 30 No. 45 03
edif 425, of. 303, Bogotá, Colombia. e-mail: [email protected]
c
Universidad Nacional de Colombia, Instituto de Ciencias Naturales, Cr 30 No. 45 03
edif 425, of. 303, Bogotá, Colombia. e-mail: [email protected]
* Corresponding author
Abstract: High mountains are harsh environments in which colder temperatures and higher levels of UV-B radiation are common. These abiotic conditions strongly affect animals` biology, often constraining their survival and reproduction. As a result, adaptations to live in such habitats are expected to evolve. Body color is thought to be adaptive to the environment that animals experience. Tegument melanization improves heat gain and provides photoprotection. Therefore, at high elevation, ectotherms are expected to be darker (well-melanized). We test this prediction in the paper wasp Agelaia pallipes (Hymenoptera: Vespidae), a species distributed across an elevational gradient in the Colombian Andes. We used Malaise traps and sampled a total of 146 wasps along nine elevations, ranging from 2,600-3,380 m above sea level. Standard digital photography was used to measure the body luminance and colour patterning in different body parts of dry-preserved specimens. There was striking variation in body luminance (darker and lighter), color patterning (patched, smoothed, homogeneous) and surface texture (shiny and matte), but the kind and degree of variation depended on the body part examined. Wasps from higher elevations had darker thoraces, confirming our prediction. Besides, at high elevation, the frequency of wasps with a matte rather than a shiny face strongly increased. Overall, our findings support the thermal melanism hypothesis and suggest that intraspecific color variation might be an adaptation to the environment of paper wasps.
Keywords: High mountain; intraspecific colour variation; thermal melanism; UV-B radiation; thermoregulation.
1. Introduction
High mountain habitats exhibit extreme abiotic conditions and animals living in such places often differ phenotypically from lowland equivalents (Hodkinson, 2005; Keller et al., 2013). The steepness of these elevational gradients is particularly relevant for analyzing the adaptiveness of intraspecific variation in species that occur from low to high elevations (Körner, 2007). Body color is thought to be adaptive to the environment that animals experience (Burtt, 1981; Cloudsley-Thompson, 1999), so that the presence of multiple colour morphs within a species is expected to broaden the range of environmental conditions under which individuals are able to survive and reproduce (Forsman et al., 2008; Caesar et al., 2010). Across animal taxa, changes in body colour according to the elevation have been described and the ultimate causes of such phenotypic clines debated (Beetles: Mikhailov, 2001; Vipers: Castella et al., 2013; Lizards: Reguera et al., 2014; Ants: Bishop et al., 2016; Law et al., 2019; Grasshoppers: Köhler et al., 2017; Crickets: Kuyucu et al. 2018). At high elevation, the most influential abiotic variables on animals` biology are the lower temperatures and the higher levels of solar radiation (Körner, 2007). In insects, cold constrains the activity levels, as these ectotherms depend mostly on extrinsic factors for thermorregulation. Additionally, UV-B light, a kind of ionizing radiation comprising a small component of the solar electromagnetic spectrum, is strongly absorbed by nucleic acids (Ravanat et al., 2001) and a number of deleterious effects have been reported for insects (Beard, 1972; Bothwell et al., 1994; McCloudx). To deal with these harsh abiotic
conditions, insects may show adaptive color variation. Eumelanin is a black pigment widespread across animal taxa and other phyla (Riley, 1997; Sugumaran, 2002; Pinkert and Zeuss, 2018). Insects at high elevation tend to have well-melanized and therefore dark (low luminance) bodies (Mikhailov, 2001; Bishop et al., 2016; Köhler et al., 2017; Kuyucu et al., 2018). Darkening the body surface improves the passive thermorregulation ability (following the thermal melanism hypothesis, Watt, 1968; Clusella-Trullas et al., 2007) and provides protection against UV-B radiation (The photo-protection hypothesis, Law et al., 2019), therefore suggesting the adaptive meaning of insect melanism at high elevation. Body melanism is supposed to be adaptive in environments other than high elevations, as it improves resistance to desiccation in drier environments (following the melanism-desiccation hypothesis, Kalmus, 1941) and provides camouflage, resistance to parasites and pathogens and protection against UV-B radiation in warmer and more humid environments (following the Gloger`s rule, Delhey, 2019). Adaptive body melanism can be detected even at a microclimatic gradient. For example, examining ant assemblages along the vertical strata in a tropical forest showed that darker ants are more common in the canopy, as these are more likely to deal with the relatively higher levels of desiccation and UV-B radiation in the canopy (Law et al., 2019). Thus, body melanism may provide a solution for multiple selective pressures. In Paper wasps (Hymenoptera: Vespidae), body colour variation is known to mediate social communication processes such as quality signaling and individual identity signaling (Cervo et al., 2015). Relatively less investigated is the possibility that wasp body colour evolved as an adaptation to environmental factors such as temperature or UV-B radiation. De Souza et al. (2017a) examined a set of dry-preserved museum species of Polistes paper wasps from different regions of the world and
established and association between thorax luminance and annual mean temperature of collection region, leading the authors to conclude that darker paper wasps have selective advantage in colder environments. Badejo et al. (2018) examined a set of ethanol-preserved Vespula vulgaris paper wasps from different climatic areas and found that higher latitudes (colder environments) may select for darker paper wasps. Both investigations (De Souza et al., 2017a; Badejo et al., 2018) support the thermal melanism hypothesis. On the other hand, body coloration of Vespa velutina paper wasps, examined by visual assessment, seems not to be related to climatic conditions (Perrard et al., 2014). To our knowledge, whether or not body coloration tracks the elevation of wasp distribution has not been tested. The swarm-founding paper wasp Agelaia pallipes have a Neotropical distribution, from Costa Rica to northern Argentina (Richards, 1978). In addition to this wide latitudinal occurrence, these wasps are often distributed from lowlands up to high mountain forests more than 3,000 m above sea level, such as in the Colombian Andes (Rodríguez-Jimenez and Sarmiento, 2008). According to Sarmiento (1993), their colonies are populous, holding up to 16,500 adult individuals. Nests are ovoid, of about 50 cm of maximum diameter, and many combs are surrounded by an envelope composed by several irregular layers. The nests can be either exposed to direct solar radiation or sheltered within trunk holes. During the day, the wasps often stay over the exposed nest surface or nearby the nest entrance, potentially receiving direct solar radiation. Rodríguez-Jimenez and Sarmiento (2008) provided anecdotal observations suggesting that these wasps seem to be darker at high elevation, but no formal assessment was performed.
Here, we investigate whether paper wasp coloration is selected by the environment at which wasps are exposed. If so, we predict that wasp coloration is associated with elevation. First, we describe intraspecific variation in the colour of different body parts in female A. pallipes. Then, we test if this phenotypic variation tracks the elevation of population distribution. From these results, we made inferences about the adaptive meaning of body colour in relation to the main abiotic selective forces at high elevation, the colder temperatures and the higher levels of UV-B light.
2. Material and Methods
2.1. Study place and wasp collection The study was performed in the Santuario Nacional de Flora y Fauna de Iguaque (5º42’21" N, 73º27’25" W, coordinates of the fourth altitude sampling site 2,850 m, close to the visitor´s center of the park). This national park is located in the eastern mountain range of the Colombian Andes, with elevation ranging between 2,400 and 3,800 m above sea level. Climate data at the site are not available but there is a strong negative relationship between elevation and temperature (R2= 0.935) and changes along the year are negligible (12.2°C - 13.6°C at 2690m) (IDEAM, 2017). The Colombian western mountain range has average temperature between 17.3°C for 2000m (12.321.0°C) and 11.3°C (5.9-15.1°C) for 3000m. The vegetation comprises oak forest, cloud forest, and paramo. Wasps were sampled by performing a set of field expeditions between March of 2000 and October of 2001. In each expedition, four malaise traps (collecting jars filled with 95% ethanol) spaced by 100 m from each other were set out for around 10 days,
after then the wasps were collected from the traps. These sampling expeditions were performed at eight different elevations, described in Table 1. Thus, the elevation of collection sites ranged between 2,600 and 3,380 m above sea level and the distances from each other between ranged between 400m and 3.2 km. Between 11 and 20 female wasps were sampled in each elevation, performing a final sample of 146 individuals across the gradient. Each wasp was removed from the ethanol, individually pinned and then set to dry eight days at room temperature (around 21°C). After then, image acquisition was performed.
2.2. Image acquisition We used standard digital photography protocols (reviewed in Stevens et al., 2007). Basically, each wasp was examined by a researcher who was blind to the collection place of each individual. This researcher captured images of four different body parts: frontal view of the clypeus, dorsal view of the mesoescutum, dorsal medial view of the second abdominal tergite and ventral medial view of the second abdominal sternite (Fig. 1 and Fig. 2). In this way, we obtained 8-bit RAW digital images by using a Leica Q2 camera adapted to a Leica S8APO stereoscope which includes a ring Leica LED illumination system at constant intensity. The stereoscope and the camera settings were kept constant (stereoscope setting: augment 4 times; camera settings: ISO = 100, f= 6,3, time of aperture = 1/160 seconds). Wasps were set at a distance of around 2 cm from the stereoscope objective. The white balance was calibrated with a 18% grey card (Kodak) and the same card was placed in the frame of each specimen as a control. We used LIGHTROOM, version 4.1 (Adobe Systems Inc.) to colourcorrect each image and them we exported them to a TIFF format.
2.3. Measuring body coloration Each picture was transferred to the Image J software, version 1.51j8 (Rasband, 2018) in order to measure the body luminance. This was performed in the RGB colour space. In this system, the luminance value of a given pixel ranges between 0 and 255, in which 0 indicates black (the darkest value) while 255 indicates white (the brightest value). The luminance of each body part was calculated as the average of eight randomly selected pixels. The mesoescutum of A. pallipes often have patterns of yellowish stripes over the black background (Fig. 2). These yellowish stripes are darker or lighter, larger or smaller, and sometimes absent, according to the wasp examined. To account for this striking variation, we performed a new set of measurements in the mesoescutum images in which we estimated the luminance of the yellowish area only, calculated as the average of four randomly selected pixels in this area. We also estimated the relative area of the mesoescutum occupied by the yellowish stripes. Of note, some wasps, especially those from higher elevations lack yellow stripes on the mesoescutum (as predicted by the Thermal melanism Hypothesis, see results). These wasps whose mesoescutum was entirely black were excluded from the analysis of the luminance of the yellowish area of the mesoescutum (31 out of 149 wasps were excluded in these analyzes). Finally, we also categorized by visual assessment wasp clypeus as shinning (Fig. 2, left) or matte (Fig. 2, right). To estimate the error associated with our luminance measurement protocol, a set of 10 wasps had the body parts measured independently by two researchers. Then, the repeated measurements were compared according to Sennar (1999). In this way, this error was 1% for both the mesoescutum and the yellow stripes, 2% for the clypeus and
5% for both the tergite and the sternite. These values are considered low (Sennar, 1999), so our measurements are highly repeatable.
2.4. Statistical analyses Descriptive statistics regarding variation in the color traits is reported as mean value, standard deviation and coefficient of variation (= standard deviation/mean). The inferential statistics was performed in different ways, as follow. To test whether there are differences in the luminance values between the different body parts (clypeus, mesoescutum, tergite and sternite) we applied the nonparametric Kruskal-Wallis test, as the assumptions required for parametric analyses were not met (even after several transformation trials). To identify which body parts are more or less luminant than the others, we applied post-hoc tests using Siegel and Castellan´s (1988) approach. To test whether luminance values in a given body part covaries with luminance values of the other body parts we applied the nonparametric Spearman`s rank correlation test. To test if the luminance of the different body parts is associated with elevation, we applied linear regression analyses. The required assumptions and outlier detection for these regressions (global fitness, skewness, kurtosis, link function, and heteroscedasticity) were previously tested using Peña and Slate`s (2006) approach based on Neyman smooth test. As a result, only the luminance values of the mesoescutum needed to be log-transformed to meet the assumptions of the regression analyses. Outliers are defined here as atypical data and not as error, these were identified also using the gvlma package approach of deletion statistics and observed in the graphic options of projections such as normal Q-Q plot, residuals vs fitted plot, half-normal Cook’s distance plot, and the
standardized residuals vs the fitted values plot (Peña and Slate, 2006; 2019); outliers were progressively removed until regression assumptions were met; outliers were identified in all variables, no more than five individuals were removed from an analysis and in no case these were clustered from a single altitude. All analyses were conducted using the software R 3.6.1. (R Core Team, 2019), using the packages Pgirmess (Giraudoux et al., 2018) and gvlma (Peña and Slate 2019). The level of significance was set at 5%. The RAW data used in this study is available as Electronic supplementary material ESM1.
3. Results
We found interindividual differences in body colour of A. pallipes (Fig. 2). Variation in body luminance was more evident for the mesoescutum (coefficient of variation, CV = 19%), followed by tergite (CV = 17%), sternite (coefficient of variation = 16%), and the clypeus (CV = 14%). The clypeus always looked homogeneously black, but there was variation in its surface texture, as some wasps had a shiny while others a matte clypeus. On the other hand, the mesoescutum may be entirely black or may had a patched coloration, as up to four stripes (0, 2 or 4) of yellowish coloration with a variable size (mean area of yellowish stripes relative to mesoescutum total area = 7%, standard deviation = 4%, CV = 67%), and luminance (Mean luminance = 95, standard deviation = 19, CV = 20%) could be present over the black background. A heterogeneous coloration was also noticed in the tergite and sternite, as both areas had a more or less smoothed pattern of yellowish and brownish areas. Thus, interindividual variation in body coloration is evident for the body surface texture (clypeus), luminance
values (clypeus, mesoescutum, tergite, sternite) and color patterning (mesoescutum, tergite and sternite). We also found differences in the luminance of the different body parts (Kruskall-Wallis test: H3 = 358.06, p = 0.001, Fig. 3A). Specifically, the darkest body part was the clypeus (Mean luminance = 53), followed by the mesoescutum (Mean luminance = 60), tergite (Mean luminance = 71) and the sternite (Mean luminance = 95). Thus, wasps are darker at the anterior (clypeus, mesoescutum) rather than the posterior (tergite, sternite) portion of the body, and they are darker at the dorsal (mesoescutum, tergite) rather than the ventral (sternite) portion of the body. Overall, the luminance between the different body parts was not correlated. The only exception was the weak positive association between the luminance of the mesoescutum and the tergite (Spearman`s rank correlation: clypeus vs. mesoescutum: r = -0.102, N = 145, p = 0.221; clypeus vs. tergite: r = -0.032, N = 142, p = 0.703; clypeus vs. sternite: r = 0.023, N = 144, p = 0.789; mesoescutum vs. tergite: r = 0.180, N = 143, p = 0.031; mesoescutum vs. sternite: r = 0.072, N = 145, p = 0.390; tergite vs. sternite: r = 0.111, N = 143 , p = 0.185). We found evidence for an association between body luminance and elevation of wasp collection, but such relationship depended on the wasp body part examined (Fig. 3B-G). Thus, the elevation of wasp collection was negatively associated with mesoescutum luminance (r2 = 0.18, b= -1.2e-04, F1,144 = 32.81, p = 0.001, Fig. 3C), but no association was detected between the elevation of wasp collection and the clypeus luminance (r2 = 0.01, b= 0.002, F1,143 = 1.08, p = 0.299, Fig. 3B), tergite luminance (r2 = 0.01, b= 0.005, F1,141 =
2.27, p = 0.134, Fig. 3D) or the sternite luminance (r2 = 0.01, b= 0.008, F1,143 = 3.51, p = 0.063, Fig. 3E). In other words, at higher elevations, wasp mesoescutum is darker, but the luminance of wasp clypeus, tergite and sternite seems not to be related to the elevation. Of note, even though the luminance in the clypeus did not track the elevation, the proportion of individuals with a matte rather than shiny clypeus dramatically increased at high elevations (Spearman`s rank correlation: r= 0.7319, N = 8, p = 0.0254, Fig. 3H). We also found that the elevation of wasp collection was negatively associated with the luminance of the yellowish area of the mesoescutum (r2 = 0.09, b= -0.02, F1,113 = 12.96, p = 0.001, Fig. 3F). Besides, as the elevation of wasp collection increases, the relative proportion of the yellowish pigmentation in the mesoescutum decreases (r2 = 0.23, b= -7,3e-5, F1,139 = 43.8, p = 0.001, Fig. 3G). Thus, at the higher elevations, the yellowish area of the mesoescutum is smaller and darker. Of note, all wasps from Lagunillas, the highest collection site, had entirely black mesoescutum as no yellow stripes were present.
4. Discussion
Agelaia pallipes paper wasps have a high degree of intraspecific variation in body colour, both at the interindividual (between individuals) and intraindividual (between different body parts) levels. Some of this variation is associated with the elevation. The thorax, specifically the mesoescutum, of A. pallipes paper wasps is darker at higher elevations. This is likely the result of changes in the colour of the yellowish
stripes over its surface as they are darker and smaller at high elevations. Darkening the body surface is often due to the deposition of melanin (Sugumaran, 2002) and this might be interpreted as an adaptation to deal with the colder temperatures at harsh environments (Valverde and Schielzeth, 2015). The thermal melanism hypothesis (Watt, 1968; Clusella-Trullas et al., 2007) proposes that darker morphs are better at converting solar radiation in heat than their brighter equivalents. Thus, darker ectothermic animals have an advantage in colder environments, because they can heat faster and become active before others. Darker morphs of several insects indeed increase in frequency at high elevations (Majerus, 1998; True, 2003; Clusella-Trullas et al., 2007). Besides, there are records in insects that the melanization related to environmental temperature often occurs in dorsal body parts that are well-exposed to direct solar radiation and are nearby the flight muscles, such as the wings of butterflies (Watt, 1968; Guppy, 1986) and the mesoescutum of other paper wasps (De Souza et al., 2017a). UV-B radiation may provoke a number of deleterious effects in insects (Beard, 1972; Bothwell et al., 1994; McCloud and Berenbaum, 1999). Thus, becoming darker at higher elevations might also be interpreted as an adaptation to deal with the increased levels solar radiation at these harsh environments, because melanin deposition on the cuticle provides photoprotection (True, 2003). The facial and abdominal coloration, although variable, was not associated with the elevation. Thus, not all variation in wasp body color tracks environmental conditions, as it has been reported for some other wasps (Perrard et al., 2014; De Souza et al., 2017a). A number of other factors might account
for this variation, such as interspecific signaling (Vidal-Cordeiro et al., 2012), intraspecific signaling (Cervo et al., 2015) or even neutral variation (De Souza et al., 2017b). The A. pallipes paper wasps exhibited darker dorsal surface and a lighter ventral surface. This countershading is a widespread pattern across animal taxa (Cuthill et al., 2016). It might have evolved due to a number of reasons, such as improvement of the passive thermoregulation ability, protection against UV-B radiation and self-shadow concealment (Caro, 2005). Thus, providing additional ways by which wasp body color might potentially be an adaptive trait. Researches often try to identify a single key function to animal external appearance (Caro, 2017), but animal coloration may experience multiple selection pressures (Lindstedt et al., 2009) in specific body parts (De Souza et al., 2017a). This might be the case A. pallipes paper wasps, as the relationship between the body coloration and the elevation of wasp collection depends on the body part examined. Thus, despite body coloration of insects and other animals is generally studied by sampling one or a few body parts, we suggest that more detailed information (getting data from many different body parts, for example, Perrard et al., 2014; Williams, 2007) might be desirable if we are to better understand the evolution of animal colour. Variation in the facial (clypeal) surface texture in A. pallipes was evident, as we could clearly assign each wasp as having a shiny or a matte face. Besides, wasps tended to be matte-faced at high elevations. All else being equal, matte rather than shiny surfaces are presumably expected to be less luminant (because shiny surfaces are more reflexive). Thus, the increase in the proportion of wasps with a matte face at high elevations could potentially improve passive thermoregulation. However, in our study, the luminance in the clypeus did not track the elevation of wasp collection even though
the frequency of matte-faced wasps improved up to 100% at the highest elevation. One possibility is that our measurement protocol was not accurate enough to capture the subtle effect the body surface texture may have had in the luminance of already well-melanized body parts. Variation in the paper wasps body coloration is complex, ranging from uniform to smoothed and patched distribution. This body coloration is often considered to be a result of cuticular pigmentation (melanin, pterin), but our findings of matte dark and shiny dark surfaces highlights for the possibility of structural coloration in paper wasps (Chavarría-Pizarro et al., 2010), this is when colour is produced not from pigments, but from the differential light reflection of the ultra‐ or microstructure surface of the cuticle (Shawkey and D`Alba, 2017).
5. Conclusion
Our study shows that the coloration of A. pallipes paper wasp varies with elevation. Concretely, thorax dorsal coloration is darker in higher elevation, presumably to improve heating of muscles associated with flight. By contrast, coloration of other parts such as head and abdomen did not vary with elevation, except for clypeus which becomes more matte with elevation. These findings suggest that wasps are darker at high elevations in order to improve their thermoregulation and protect them from UV-B radiation
6. Acknowledgements Authors thanks members of the Laboratorio de Biología Comparada de Insectos of the Universidad Nacional de Colombia for their support during data gathering. This
work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (grant number 2015/05302-0 to AR de Souza) and by the National University of Colombia Hermes project 8367 (support to CES and AZM).
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Figure Legends
Table 1. Details of field expeditions to collect A. pallipes paper wasps in different elevations. Fig. 1. Sketch of wasp body indicating parts in which colour variation was examined. Fig. 2. Examples of body colour variation in A. pallipes paper wasps from different elevations. Pictures illustrate some variation in the female clypeus (although consistently black, it ranges from matte to shiny as the elevation increases, comprising differences in surface texture), mesoescutum (note the patched coloration and the variation in the number, size, and luminance of the yellowish stripes, so that they are all reduced as the elevation increases), tergite and sternite (note the variation of brownish and yellowish smoothed patterns). Fig. 3. Body colour variation in A. pallipes and its relation with the elevation of collection. In A, within-box lines shows the median, the whiskers show the minimum and maximum values and different letters indicate statistical differences between the
classes. From B to G, each black dot refers to a single individual and the lines represent a significant association.
Electronic supplementary material ESM1. RAW data used in this study.
Collection site Cabaña Chaina El Nispero Site three La Planada and Cabaña Carrizal Site five Cerro Pan de Azúcar Quebrada Carrizal Lagunillas
Elevation (meters above sea level) 2.600 2.730 2.795 2.850 2.975 3.300 3.350 3.380
Time of collection May/2001 Oct/2001 Mar/2000 Jan/2001 and Oct/2001 Mar/2000 Mar/2001 Jan/2001 Mar/2001
• High mountains exhibit harsh abiotic conditions, like low temperatures and higher levels of UV-B radiation • Tegument melanization (darkening) improves heat gain and provides photoprotection • Females of Agelaia pallipes are darker at high elevations • Intraspecific colour variation might be an adaptation to the environment where of paper wasps live.