Characterization of Cissia hermes Phenotypes in Costa Rica Kerry Piper Department of Ecology and Evolutionary Biology University of California, Los Angeles EAP Monteverde Tropical Biology Program, Spring 2006 9 June 2006 ABSTRACT: Variation within Cissia hermes (Satyrinae, Nymphalidae, Lepidoptera) manifests itself in the wing size and ocelli size, among other phenotypic characters. In this study, I quantified the differences between individuals and evaluated it with respect to the time and location of specimen collection. The hind wing area and ocelli area of 88 individuals of C. hermes were analyzed using Adobe Photoshop CS2 (Adobe Systems, San Jose, California, USA). Individuals were obtained from 36 sites in Costa Rica and two sites in Panama representing temporal and spatial variation. I found wing area to vary among season and drainage (Caribbean and Pacific) but not habitat or elevation. I found that the area of ocelli increases in the rainy season, wet habitat, and low elevations. Each of these variables corresponds to decreased levels of photoperiod providing a causal link between the size of ocelli in C. hermes and this factor. RESUMEN: La variación dentro de Cissia hermes (Satyrinae, Nymphalidae, Lepidoptera) se manifiesta en el tamaño del ala y tamaño de ocelos, entre otros caracteres fenotipito. En esta investigación, cuantifiqué las diferencias entre individuos y las evalué con respecto al momento y ubicación de la colección del espécimen. Analicé el área de los ocelos basales y el área del ala de 88 individuos de C. hermes utilizando Adobe Photoshop CS2 (Adobe Systems, San Jose, California, EEUU). Los individuos fueron obtenidos de 36 sitios en Costa Rica y dos sitios en Panamá que representan la variación temporal y geográfica. Encontré que el área del ala varió entre estaciones y la vertiente (Caribe y Pacífica) en que fue colectadas pero no hábitat o elevación. Encontré que el área de los ocelos aumentó en la estación lluviosa, el hábitat húmedo, y las elevaciones bajas. Cada una de estas variables corresponde a niveles en los que el fotoperiodo presenta una disminución, brindando una unión causal entre el tamaño del ocelo en C. hermes y este factor.

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Nature abounds with variation. Human fingerprints are unique for every individual as are the stripes of a zebra and the veination of honey bees (Cummins 1961, Klingel 1974, and Kastberger 2003). Differences in the wing patterns of butterflies facilitate the classification of the 12,000 described butterfly species in the world (McMillan 2003). Wide variations in wing patterns not only exist between members of related species but also within members of the same species (Brakefield 1999). Thousands of overlapping scales, each containing a single pigment of color, underlie the wing patterns of butterflies (Nijhout 1991). Patterns, such as the eyespot or ocellus, are created by groupings of pigments. In the development of an ocellus, pigments are deposited around a focus located midway between wing veins (Nijhout 1991). The focus then signals the surrounding cells to differentiate into rings composed of colored scales. (McMillan 2003). The nymphalid ground-plan, which dictates the location and form of wing characters, is controlled by genetics (Nijhout 1990). Environmental factors influence the expression of the genes (Scott 1986). Photoperiod, temperature, seasonality, and vegetation are all environmental factors associated with variation in wing pattern (Brakefield 1999, DeVries 1987). Cissia hermes, a member in the subfamily Satyrinae, exhibits seasonal variation. Dry season forms may have the ventral pattern washed out with ocelli reduced to black dots (DeVries 1987). The aim of this study was to investigate the effects of temporal and spatial variables on the wing area and ocelli area of C. hermes. The effect of month and season as temporal variables were investigated. Spatial variables of habitat type, elevation, and drainage were also investigated to illustrate the environmental factors affecting the variation in ocelli size. NATURAL HISTORY. –Cissia hermes is a member of the family Nymphalidae and subfamily Satyrinae, a group known as the forest nymphs. It has a forward wing length of 16-20mm with a bubble-like formation at the base of the forewing vein. The ventral side of the hind wing has wavy medial lines and a row of six border ocelli. The ocelli have single grey pupils ringed in black, yellow, and then brown (Figure 1). C. hermes is a widespread and common butterfly in Costa Rica as described by DeVries (1987). The average lifespan is though to be two to three months with populations constant throughout the year. The hostplants are Poaceae and thus it can be found in pastures, open areas, roadsides, and forest edges. The range of this small butterfly extends from Southern United States throughout the neotropics. Populations are persistent from 0-1500m. METHODS: STUDY SITES. –I sampled 88 specimens of C. hermes from 36 sites in Costa Rica spanning all seven provinces as well as two sites in Panama in the Chiriquí Province (Table 1). I captured specimens with a butterfly net in the transition period between the dry and rainy seasons (9-12 May 2006) from the Monteverde Region, including Tornos, Cerro Plano, Monteverde, and San Luis Abajo. I identified these sites due to the large grass coverage and direct sunlight present. I then pinned and mounted these specimens with wings spread. Specimens from other regions were obtained from the entomology collection at the Department of Natural History at the

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Costa Rican National Museum. Specimens from every month of the year are represented in the collection and were collected primarily in the 1970’s and 1980’s. The diverse habitats of Costa Rica are represented in the many sites from which C. hermes were collected (Table 1). Costa Rica, even as a small country at 51,100km2, contains 19 closely stacked life zones. The climactic differences in the country, explained by the habitat and topography of the region, were used in this study to tease apart the variation of C. hermes. The rainy season generally spans from May to December with the Atlantic side trailing the Pacific side (Coen 1983). I therefore clustered the months of June to November as rainy season, January to April as dry season, and May and December as transition period for the purposes of this study. Additionally, some locations experience greater amounts of rainfall in a given year than others (Coen 1983). For example, Guanacaste receives far less rain than the Osa Peninsula. To measure this variable, I considered locations with more than three meters of annual rainfall to be wet habitats and those with fewer than three meters to be considered dry habitats. The elevation of Costa Rica ranges from sea level to 3,810m. As the elevation increases, the difference between mean maximum and minimum monthly temperatures decreases as does the amount of sunlight in a day, or photoperiod (Coen 1983). I used ranges of 200m to analyze the effect of elevation on the wing characters. The Atlantic and Pacific slopes differ in the patterns of temperature and rainfall (Coen 1983). For this reason, the slopes are analyzed separately as an additional means to characterize the variation in ocelli area of C. hermes. CHARACTERIZATION. –With the wings spread, I laid each butterfly against a background of quadralined graph paper. I then took a photograph of the dorsal side using a Nikon Coolpix 5200 camera which I imported into Adobe Photoshop CS2 (Adobe Systems, San Jose, California, USA). This powerful program is a valuable tool not only for editing, but also analysis of photographs. Using it and the graph paper as a guide, I cropped each photograph to a 4cm by 4cm square with the perspective option enabled. I used this option to correct for any distortions resulting from the angle in which I took the photograph. I then developed a factor specific to each photograph to convert from pixels to area. The expanded Histogram and Information windows displayed the number of pixels and dimension from two arbitrary rectangular selections I made on each photograph. I used conversion factor  # pixels  Χ    width × height  to determine the area of organic shapes such as the hind wing and ocelli. I selected the entire hind wing analyzed using the Lasso tool. This tool enabled me to include any wing area that may have been damaged from predation attempts or natural wear while I drew the outline of the hind wing to select it. I systematically quantified the size of the black ring in the second ocellus on the ventral hind wing (Figure 1) using Photoshop. The six ocelli on the basal wing covary (personal observation). As the second ocellus is the largest of these, I chose it as an indicator of total ocelli size. I selected the ocellus with the Magic Wand tool using a tolerance set to 25 pixels. As tolerance determines the similarity or difference of pixels selected, this value enabled the selection of the entire black area of the ocelli without selecting additional pixels outside of this wing character. I recorded and converted the number of pixels of each selection to area by multiplying each with the conversion factor corresponding

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to the photograph. The “ocelli index,” used to express the relationship between the two characters, is the blackened area of the second ocellus as a percent of the total hind wing. ANALYSIS. –I conducted all analyses using JMP (SAS Institute Inc. 2000). The relationship between wing area and ocelli area was examined using a linear regression. Relationships among wing characters and temporal and spatial variables were examined using ANOVA and the Student T-test. RESULTS: WING CHARACTERS. – Wing area and ocelli area do not covary in the 88 butterflies I sampled (R2=0.036, F=3.1912, p=0.0775) (Figure 2). All variables examined in this study showed a relationship with the ocelli size. TEMPORAL VARIATION. –Variation exists between the months in which the butterflies were collected over all wing characters: wing area (R2=0.3755, F= 4.1000, p<0.0001), ocelli area (R2=0.3699, F=4.0020, p<0.0001), and ocelli index (R2=0.3259, F=3.2958, p=0.0010) (Figure 3). When months are grouped into seasons, the effects are more evident (Figure 4). Eighteen individuals were collected in the dry season, 29 in the wet season, and 40 in the transition period (Table 1). Through this analysis, the seasonal variation of wing characters in the butterflies collected is as follows: wing area (R2=0.2150, F= 11.5058, p<0.0001), ocelli area (R2=0.2476, F=13.8248, p<0.0001), and ocelli index (R2=0.1853, F=9.5533, p=0.0002) (Figure 4). Wing area of dry season specimens did not vary with those of wet season specimens (Abs(dif)-LSD=-0.06831). The size of ocelli varied across these two seasons with dry season specimens bearing smaller ocelli than wet season specimens (dry=0.01209cm2, SDdry=0.00843; rainy=0.01681cm2, SDrainy=0.007312; Abs(dif)-LSD=0.00067). Butterflies in the transition period interestingly exhibit both the lowest wing area (transition=1.1958cm2, SDtransition=0.2187) and ocelli area (transition=0.008112cm2, SD=0.005442). They also have the lowest standard deviation in both of these characters. SPATIAL VARIATION. – When wing area and ocelli area are analyzed as functions of spatial variables, an interesting trend is observed. There is no significant difference in the wing area of butterflies in either habitat type or elevation (Figure 5a, 6a). However, the area of the ocelli varies in relation to each of these variables (Figure 5bc, 6bc). In addition, wing area of butterflies varied in relation to drainage (Figure 7a), while ocelli area and ocelli index did not (Figure 7bc). I analyzed 60 specimens from a dry habitat and 28 specimens from a wet habitat (Table 1). No variation is evident in the wing area (R2=0.001327, F=0.1143, p=0.7361). However, variation is evident in the ocelli area (R2=0.1930, F=20.5695, p<0.0001) and the ocelli index (R2=0.2216, F=24.2813, p<0.0001) (Figure 5). The area of ocelli in butterflies from dry habitats (dry=0.009533, SDdry=0.006308) is reduced in comparison to the wet habitats (wet=0.01674, SDwet=0.008148).

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Butterflies in this study represent nine elevation ranges from sea level to 1,800m (Table 1). Again, no variation exists between the wing area of butterflies from different elevations (R2=0.1515, F=1.7631, p=0.0970), but does exist in both the ocelli area (R2=0.0970, F=5.7653, p<0.0001) and ocelli index (R2=0.3566, F=5.4938, p=<0.0001) (Figure 6). The elevational differences in ocelli area show that butterflies in lower altitudes have larger ocelli. Twenty two specimens originated from the Caribbean slope while 66 specimens originated from the Pacific slope. Wing area was the only character to exhibit variation (wing area: R2=0.05717, F=5.2146, p=0.0249; ocellus area: R2=0.000031, F=0.0026, p=0.9593; and ocellus index: R2=0.001902, F=0.1639, p=0.6866) (Figure 8). C. hermes hind wings from the Caribbean slope (Caribbean=1.4199, SDCaribbean=0.2086) were slightly larger than those from the Pacific slope (Pacific=1.2834, SDPacific=0.2585). DISCUSSION: Wing area cannot account for the variation in ocelli area since the two characters do not covary. Therefore, other variables must account for this variation. Interesting relationships found between the variables studied uncover the root of variation in C. hermes. Marked differences in vegetation, rainfall, and temperature are present between the wet and dry seasons (Coen 1983). The availability of food as the larva develops could explain the differences in wing size between seasons. The low variation in wing area and ocelli area of butterflies in the transition period is likely explained by the great number of specimens obtained in Monteverde during this time. Any temporal differences present within the transition period are masked by this fairly homogeneous population. Costa Rica is a small country. Natural selection may not act on wing area between sites from different habitats or elevational ranges over such short distances. However, populations from the two drainages do exhibit a difference in wing area. It is difficult for organisms to cross tropical mountain ranges, limiting the ability of populations to interbreed (Coen 1983). Genetic drift over time may account for the difference in wing area between the Pacific and Caribbean slopes. Area of the ocelli did not vary across the two drainages, as this analysis homogenizes all other variables studied which do vary. Season, habitat, and elevation are affected by photoperiod and temperature. Photoperiod is highest in the dry season and in dry habitats as is the temperature (Coen 1983). Ocelli are smallest in the dry season and in dry habitats (Figure 3b, 4b). Photoperiod increases along an elevational gradient while temperature decreases (Coen 1983). Ocelli are smallest in higher altitudes (Figure 5b). Therefore it can be synthesized that photoperiod and not temperature is responsible for the size of ocelli in C. hermes. Controlled laboratory experiments such as those detailed by Brakefield (1999) should be conducted to verify this hypothesis. Photoperiod acts on the variation of ocelli size in many cases of seasonal polyphenism such as Precis coenia (Brakefield 1999). The endocrine system, which takes part in the regulation of ocelli size, is influenced by environmental factors through the use of ecdysteroids in several species studied (Roundtree 1995, Koch 1996, and Brakefield 1998). In the case of Bicyclus anynana, the ocelli of which are influenced by temperature, these steroids act between

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28-48 hours after pupation yielding a wet season form if an early peak in ecdysteroids is present and the dry season form if delayed (Brakefield 1999). The ecdysteroids act on the expression of five to six genes implicated in the variation of eyespot size (Brakefield 1996). Natural selection is able to operate on these genetic differences between morphotypes. It works strongly on wing pattern due to the need of butterflies to avoid predators’ attacks, blend in with the flora, and attract potential mates (Brakefield 1999). In the case of ocelli, the ability to avoid predator’s attacks works in an inverse relationship with the butterfly’s ability to blend in with the flora. Bengston (1981) found that when predation pressures are high on Maniola jurtina, ocelli function to deflect the attacks of predators away from vital body parts. However, he noticed when predation pressures are lower, the ocelli may hinder the ability of the butterfly to remain cryptic. Such a pressure seems likely in Bicyclus anynana in which ocelli are reduced or absent in the dry season form, in conjunction with a lowered activity level during this time (Brakefield 1999). Though the activity level of C. hermes was not a part of this study, it is possible that the reduced size of ocelli may allow the butterfly to remain cryptic as well. Thus determination of the root of variation in C. hermes, presumably photoperiod in regard to ocelli area, can shed light on the selection pressures faced by it. ACKNOWLEDGEMENTS: Thank you to Dr. William Haber for sharing his knowledge of butterfly variation with me. Many thanks to the Department of Natural History at the Costa Rican National Museum in San Jose for allowing me to photograph their collection of Cissia hermes. Thank you to the people of Tornos who graciously assisted me in my search for my species and allowed me to collect butterflies on their farms. Sincere thanks to the Arroyo Solis family for their hospitality during my stay in Cerro Plano. Thanks to all the Bio Staff who have assisted me from the formulation of my research question through the development of my procedure and interpretation of my results, notably: Ruth for accompanying me to San Jose; Fede, my second reader, for offering the suggestions needed to polish my paper; Ramsa, my primary advisor, for encouraging me to appreciate my data during my initial frustrations and supporting me throughout the research process; and Frank for suggesting I analyze additional variables, proving many useful contacts without which this research would not have been possible, and making this experience possible. LITERATURE CITED: Bengston, S. A. 1981. Does bird predation influence the spotnumber variation in Maniola jurtina? Biol. J. Linn. Soc. 15: 23–27. Brakefield, P. M., and V. French. 1999. Butterfly wings: the evolution of development of colour patterns. BioEssays. 21:391-401. -----F. Kesbeke, and P. B. Koch. 1998. The regulation of phenotypic plasticity of eyespots in the butterfly Bicyclus anynana. Am Nat. 152: 853–860. -----J. Gates, D. Keys, F. Kenseke, P. J. Wijngaarden, A. Montelro, V. French, and S. B. Carrol.

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1996. Development, plasticity and evolution of butterfly eyespot patterns. Nature. 384: 236-242. Coen, E. 1983. Climate. In D. H. Janzen (Ed.). Costa Rican Natural History. pp. 35-45. University of Chicago Press: Chicago. Cummins H., Midlo C. 1961. Fingerprints, Palms and Soles: An Introduction to Dermatoglyphics, Dover Publication, Inc New York. DeVries, P. J. 1987. The Butterflies of Costa Rica and Their Natural History. Princeton University Press: New Jersey Kastberger, G., S. Radloff, and G. Kranner. 2003. Individuality of wing patterning in Giant honey bees (Apis laboriosa). Apidologie, 34: 311-318. Klingel H. 1974. Soziale Organisation und Verhalten des Grevy-Zebras (Equus grevyi), Z. Tierpsychol. 36, 37–70. Koch, P. B., P. M. Brakefield, and F. Kesbeke. 1996. Ecdysteroids control eyespot size and wing color pattern in the polyphenic butterfly. Bicyclus anynana. J Insect Physiol. 42:223–230. McMillan, W. O., A. Monteiro, and D. D. Kapan. 2003. Development and evolution on the wing. TRENDS in Ecology and Evolution. 17(3): 125-133 Nijhout, H. F. 1991. The Development and Evolution of Butterfly Wing Patterns, Smithsonian Institution Press ----- 1990. A comprehensive model for colour pattern formation in butterflies. Proc. R. Soc. Lond. B 239: 81-113. Paulsen, S. M. 1994. Quantitative Genetics of Butterfly Wing Color Patterns. Developmental Genetics. 15:79-91 Roundtree D. B., and H. F. Nijhout. 1995. Hormonal control of a seasonal polyphenism in Precis coenia (Lepidoptera: Nymphalidae). J Insect Physiol. 41:987– 992. Scott, J. A. 1986. The Butterflies of North America: A Natural History and Field Guide. Stanford University Press: California