Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 1 D iversity and p henotypes of diurnal and nocturnal c oleopterans in the Monteverde cloud f orest zone Marianne Kim Department of Ecology and Evolution University of California, Santa Cruz EAP Tropical Biology and Conservation Program, Spring 2016 16 December 2016 ABSTRACT Coleoptera, or more commonly known as beetles, are the largest and most diverse order within the arthropods. This study aimed to categorize the diversity between diurnal and nocturnal beetles as well as determine if there are dis tinguishing phenotypes between the two groups. For a total of five days, I collected diurnal and nocturnal beetles throughout the tropical cloud forests of the Monteverde zone in Costa Rica and identified to Family. For each species, I recorded the size, c oloration (number of colors visible and light versus dark tones), pattern, texture, and iridescence and compared between diurnal and nocturnal beetles. With respect to diversity, my results revealed that while diurnal beetles are more divers e within the le af beetle family, Chrysomelidae nocturnal beetles have the highest diversity within the scarab beetle family, Scarabaeidae indicating that diurnal or nocturnal activity may be more correlated the taxonomic category than to individual species phenotypic traits. This is further supported by the presence of exclusively diurnal as well as exclusively nocturnal families in my data T test analysis proved size to be not correlated with beetle diurnal or nocturnal activity. The only phenotypic significant difference I discovered was that nocturnal beetles are darker in coloration than diurnal beetles, indicating that the darker tone is possibly an adaptation for nocturnal beetles to remain hidden while active in the darkness of the night. Other phenotypes may instead show more correlation to crypsis, aposematism, and sexual selection than diurnal and nocturnal activity. Diversidad y fenotipos de colepteros d iurnos y n octurnos en el b osque nuboso de la z ona de Monteverde RESUMEN El Orden Coleoptera contiene los comnmente conocidos como escarabajos y son el grupo ms grande y diverso dentro de los artrpodos. El objetivo de este estudio fue categorizar la diversidad entre los escarabajos diurnos y nocturnos y determinar si hay fenotipos distintivos correspondientes a cada grupo. Durante un total de cinco das, colect escarabajos diurnos y nocturnos en los bosques tropicales de la zona de Monteverde en Costa Rica y los identifiqu a nivel de Familia. Para cada especie, registr el tamao, la coloraci n (en dos categoras: nmero de colores visibles, tonos claros y oscuros), los patrones, la textura y la iridiscencia y compar estos datos entre escarabajos diurnos y nocturnos. Con respecto a la diversidad, mis resultados revelaron que los escarabajos d iurnos presentan mayor diversidad dentro de la Familia Chrysomelidae, mientras que los escarabajos nocturnos tienen la diversidad ms alta con la familia Scarabaeidae, indicando que la actividad diurna o nocturna puede estar ms correlacionada con su categ ora taxonmica que con rasgos fenotpicos independientes. Este
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 2 resultado es apoyado por el hecho de que hubo familias exclusivamente diurnas o exclusivamente nocturnas en mis datos. Una prueba de T student demostr que el tamao no est correlacionado co n la actividad diurna o nocturna de los escarabajos. La nica diferencia fenotpica que descubr fue que los escarabajos nocturnos son de coloracin ms oscura que los escarabajos diurnos, lo que indica que el tono ms oscuro podra ser una adaptacin para que los escarabajos nocturnos permanezcan ocultos mientras estn activos en la oscuridad de la noche. Otros fenotipos pueden mostrar ms correlacin con la cripsis, el aposematismo y la seleccin sexual que la actividad diurna y nocturna. INTRODUCTION In the animal kingdom, the class that occupies more than 75% of the popul ation is Insecta (Daly 1978 ). Within this large group, the most diverse grou p of insects is the Coleoptera O rder, or more commonly known as the order of the beetles. Beetles undergo a complete metamorphosis, meaning that they morphologically change as they grow from eggs to larvae to adults (Crowson 2013) Beetles occupy a wide range of habitats ranging from within plant material or rotting wood to carrion or dung. Most are terrestria l but there are some species are known to be are aquatic Features that characterize these organisms are compound eyes, compact bodies mandibles designed for chewing, and sclerotization of an ex oskeleton (White 1983) T he key feature that separate beetles from other orders are the modified front pair of wings that form the elytra. main function is to prot ect the membranous wings that are used for flight. In most beetles, the elytra extends to the end of the abdomen, but in some coleopteran fam ilies, the elytra goes past the abdomen or only covers the thorax. Most of beetle coloration, design, and shape, come from the elytra and helps in identification of different species. Like other animals, different species of beetles are active at differen t intervals of the day. While majority of beetles are diurnal meaning that they come out during the hours of sunlight, there are select nocturnal species that are active when the sun disappears and their world is encased in darkness (Crowson 2013) One of the main factors why most species are diurnal is that they require the sun to stay active (Rosenthal 2006) Whether it is because it supplies them with thermo energy to forage, or allows them to see more clearly, the sun is an important component in contro lling diurnal activity. Nocturnal species are active during times of colder tempera ture and darkness. These conditions are best for organisms with the ability to see in the dark or those with other heightened senses that help them to maneuver while with po or sight. There are also few species that are crepuscular or active during twilight hours between day and night or vice versa. Crepuscular species are awake during the in between hours either because their prey also come out during this time or to forage before other competitors are awake (T hery 2008) With about 750 ,000 known species of insects, 4 00,000 of them are beetles (Crowson 2013) In the tropical forest s of Costa Rica there are 110 out of the 178 identified families of beetles ( Nadkarni 2000) The Monteverde zone is especially species abundant in animals including beetles. Monteverde is home to both the largest beetle in the world, the Hercules beetle of the family Scarabaeidae as well as the smallest beetle in the world, the beetle of the family Ptilidae Beetles are extremely diverse in their morphology and
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 3 behaviors that are characterized by different adaptive significance. Rosenthal (2006) sampled the abundance and diversity of diurnal and nocturnal insects of the montane rainforest in Monteverde, Puntarenas, Costa Rica. He discovered that the biodiversity of diurnal insects greatly surpasses that of nocturnal ones and hypothesized that it may be due to the additional es of the night than the warmer ones of the day this includes the need to use s tored energy to be active at night as opposed to during the day when the heat from the sun helps regulate body temperature. Immense di versity amongst beetles inspired me to propose two question s : What is the diversity of diurnal and nocturnal Coleoptera in the Monteverde zone cloud forests and are there distinguishing phenotypes between the two groups of beetles? MATERIALS AND METHODS During a total of five days, I collected beetles at three different locations within the cloud forests of Costa Rica Two locations were in Monteverde, Puntarenas: the trail to the TV towers near the Estacin Biologica w ith the highest elevation of 1575 meters above sea level and the Crandell Memorial Reserve behind the Instituto Monteverde at 1,300 meters above sea level The third collection site was the lowest elevation at 900 meters above sea level and San Gerardo an area within the protect ed reserve Bosque Eterno de los Nios To avoid collecting beetles that are crepuscular, or active in the twilight hours, for diurnal beetles I only used specimens found between 9 am and 4 p m for diurnal beetles and between 6 pm and 10 pm for nocturnal beetles I collected for about three hours in the day and three hours at night, estimating about 15 total hours of searching in the day as well as at night I actively caught all specimens, either with an aspirator, plastic bag, plastic container, or with my hands. I determined beetles as active if they showed movement or were exposed to the environment I avoided digging through soil or rotten wood, which are known habitats of many beetles, because I wanted to avoid capturing beetles that were inactive at that time of collection. With minimum disruption, I searched on and under all substrates of the forest. After collection, I stored the beetles in a freezer The cold temperature human e ly killed the specimens to later identify them Smaller beetles were le ft inside for a minimum of one hour while larger ones were inside for at least three hours. After an adequate amount of time in the freezer, the beetl es were pinned. Once the beetles were pinned, they were categorized by the time of day they wer e active: d using A field guide to the beetles of North America by R. E. White Then, I analyzed and categorized the following phenotypic characteristics: size, coloration, patterns, textures, and iridescence. S ize was measured in millimeters from the tip of the eyes to the end of the abdomen. Some species have elytras that extend p ast their abdomen while others such as weevils, have elongated snouts. Doing so ensured consistency in measuring beetle size. Other phenotypes were analyzed from only the dorsal side of the beetles since majority of the time, the underside of beetles are unexposed.
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 4 Coloration of coleopteran was split into two separa te characteristics: number of colors visible and dark versus light coloration I counted the number of colors easily visible on each species and categorized them as having one color, two colors, or three or more colors. To separate the species between ligh t or dark colors, I held the beetles against a white background and then against a black background. If they were more noticeable against the light background they were placed in the dark category and vice versa. Patterns were narrowed down to five diffe rent categories: no pattern, spot s or blotches, speckles, stripes, or blocks. No pattern was present when beetles held only one color I categorized spots and blotches as when multiple sections of the elytra had different coloration than majority of the el ytra Speckles were miniature dots of different colors that are difficult to differentiate. I indicated blocks of color when only two colors were visible in large distinct sections. I created three categories of texture: glossy, smooth, and rough. A gloss y texture holds reflectance on the surface of the beetle while a rough texture holds little to none. The smooth surface is the intermedi ate of the two other groupings. I determined texture visually rather than through touch. Using the same categories of i ridescence as Seago (2008) I separated the beetles by those without iridescence, multilayer refractors, three dimensional photonic crystals, and diffraction of vision is varied meaning that different colors are visible at different angles ( Mason 1927 ). Multilayer refractors are the most common of the three types and this allows you to see two or three different hues. Photonic crystals are usually reserved f or weevils that have ir idescent miniature scales and require microscopic lenses to see its full effect. Diffraction gratings are present when you can see the full spectrum of white light red, orange, yellow, green, blue, violet Iridescence is the only phenotype I observed using a dissecting scope. RESULTS A total of 69 different species in 15 families were collected: 38 diurnal, 28 nocturnal, and three species seen in both times of the day (Fig 1). Because the sample size found to be both diurnal and nocturnal was so small, I decided to remove this group from data analysis. Majority of the diurnal species belonged to the leaf beetle family, Chrysomelidae (n =18). I found the most nocturnal species of the scarab beetle family, Scarabaeidae (n =8). Families that were strictly diurnal were Coccinellidae, Lycidae, and Tenebrionidae while Carabidae, Cleridae, Lampyridae, and Ptilodactylidae only held nocturnal species.
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 5 Figure 1: Number of species collec ted per f amily of Coleoptera and their time of day activity Although the average sizes between diurnal and nocturnal beetles were similar (with diurnal beetles averaging slightly larger) the standard deviation shows that sizes between the species varied greatly ( Fig 2 ). Further t test analysis showed that size is insignificant independent of diurnal or nocturnal activity ( t = 0.11 df = 64, p = 0.91 ). Figure 2 : Average sizes of diurnal and nocturnal beetles In some cases, I found multiple individuals of a specie s so I calculated the species average size before calculating diurnal and nocturnal averages 2 0 1 18 0 1 5 1 0 0 4 0 4 1 1 1 3 1 2 2 0 6 1 0 1 0 2 8 1 0 1 1 1 0 2 4 6 8 10 12 14 16 18 20 Number of Species Families Dirunal Nocturnal Both 8,86 8,62 0 10 20 30 Diurnal Nocturnal Size (mm)
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 6 Nocturnal beetles had a higher frequency of single colored species while diurnal species had a higher frequency of having two colors (Fig 3 ). Chi squared analysis proved number of colors visible on beetles to be unrelated to diurnal or nocturnal activity ( X 2 = 0.93, df = 65, p = 0.63 ). Figure 3 : Frequency of total number of colors visible on diurnal and nocturnal beetles I found that while the diurnal species were about evenly split between light and dark coloration, nocturnal species held more dark tones than light tones (Fig 4 ). Out of 28 nocturnal species, 22 of them had darker colors (Fig 4 ) Statistical analysis showed light versu s dark coloration to be significant to diurnal and nocturnal activity ( X 2 = 4.69, df = 65, p = 0.030 ) Figure 4 : Frequency of light coloration and dark coloration in diurnal and nocturnal beetles. While diurnal beetles had a higher frequency of species displaying patterns, nocturnal beetles were about 50/50 between species with patterns and species without patterns (Fig 5 ). Of the nocturnal species that did hold patterns, the most frequent pattern w as speckles (18%). The 0.42 0,5 0.47 0.36 0.11 0.14 0% 20% 40% 60% 80% 100% Diurnal Nocturnal Frequency 1 Color 2 Colors 3 Or More Colors 0.47 0.21 0.53 0.79 0% 20% 40% 60% 80% 100% Diurnal Nocturnal Frequency Light Dark
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 7 type of pattern that occurred most on diurnal beetles was either the stripes or blocks. (18%). A Chi squared test proved presence of a pattern to be insignificant to diurnal or nocturnal activity ( X 2 = 0.85 df = 65, p = 0 .36 ) Figure 5 : Frequency of different patterns on diurnal and nocturnal beetles Majority of species had a glossy texture as opposed to smooth or rough ( Fig 6 ). Nocturnal beetles had proportionally less reflective texture t han diurnal beetles. Statistical ana lysis proved texture of beetles to be insignificant to diurnal or nocturnal activity ( X 2 = 3.44 df = 65, p = 0 .18 ) Figure 6 : Frequency of different textures visible on diurnal and nocturnal beetles. Most of the specimens did not display any iridescence (Fig 6). Of those that did, the most frequent form of iridescence was multilayer reflectors. Diffraction gratings were seen on only one specie s in Scarabaeidae which belonged to the nocturnal group. Statistical analysis showed iridescence to be independent o f diurnal or nocturnal behavior ( X 2 = 0.076, df = 65, p =0.78 ). 0.42 0.54 0.16 0.14 0.05 0.18 0.18 0.07 0.18 0.07 0% 20% 40% 60% 80% 100% Diurnal Nocturnal Frequency No Pattern Spots/Blotches Speckles Stripes Blocks 0.66 0.43 0.21 0.36 0.13 0.21 0% 20% 40% 60% 80% 100% Diurnal Nocturnal Frequency Glossy Smooth Rough
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 8 Figure 7 : Frequency of different iridescence in diurnal and nocturnal beetles DISCUSSION The largest sample size came from the diurnal leaf beetles, Chrysomelidae co ming to the conclusion that diurnal beetles have the highest species richness in the family of leaf beetles On the other hand, nocturnal beetles may have their highest richness among scarab beetles, Scarabaeidae because nocturnal scarab beetles were the secon d largest group collected This information and the d iscovery of families that were exclusively diurnal or exclusively nocturnal also led me to believe that taxonomic category may have more correlation to time activity of beetles than their phenotypic char acteristics meaning that species in certain families evolved together to be diurnal or nocturnal The t test of average size between diurnal and nocturnal beetles revealed that there is no correlation between beetle size and diurnal versus nocturnal act ivity. When the t test result is removed from the analysis, the average size distribution between the diurnal and nocturnal beetles shows that they are about similar in size. However, the large standard deviation of the diurnal beetles indicates that this average is flawed due to a large outlier a diurnal beetle in the longhorn family ( Cerambycidae ) Huffaker and Gutierrez (1999) stated that nocturnal insects tend to be larger than diurnal insects due to the lack of sunlight during their time of activity Diurnal insects rely on the sun to regulate the temperature of their bodies as they forage in the day. Nocturnal insects on the other hand must be well adapted for thermoregulation if active during colder nocturnal temperatures. Therefore, my t test analy sis of the average sizes of diurnal and nocturnal beetles may have inaccurately determined size to be independent of diurnal or nocturnal activity due to the small sample size of 69 species. 0.61 0.57 0.34 0.29 0.05 0.11 0 0.04 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Diurnal Nocturnal Frequency No Iridescence Multilayer Reflectors 3D Photonic Crystals Diffraction Gratings
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 9 After performing Chi squared tests on the remaining phenotypes, the only phenotype that showed significance was the light versus dark coloration. The results of the test revealed that darker coloration occurs more often than lighter coloration in nocturnal beetle s as opposed to diurnal species which are sp lit about 50% in each coloration I hypothesize that dark coloration is an evolutionary adaptation for nocturnal beetles to stay hidden from predators against the darkness of the night. Additionally dark coloration can also be an adaptive characteristic o f beetles that are themselves predators and need to be unnoticeable when they hunt Beetles that are diurnal hunters tend to be more metallically colored while nocturnal ones are usually black reinstating that they are better adapted to be darker colored ( Crowson 2013 ). If diurnal and nocturnal activity is independent of phenotypes such as quantity of colors patterns textures and iridescence other defense mechanisms may be the purpose of such diversity For example, aposematism, or w arning colorations, that certain insects display allow them to avoid being eaten because v ibrant colors and unique patterns tell predators that they are poisonous or distasteful (Jones 1932) Better crypsis in the forest is another defense property that ma y influence the phenotypic differences amongst beetles. Iridescence for example give beetles crypsis against the green foliage because green is the most abundant color seen in iridescence (Parker 1998) S exual selection is another determinant of different phenotypes among beetles. Bezzerides et al ( 2007) discovered that the presence of re d coloration, or less black coloration, on Asian lady beetles, Harmonia axyridis is linked to higher alkaloid levels which are chemical defenses against predators. His discovery along with prior studies stating that Harmonia axyridis with more red coloration or less black coloration have higher mating probabilities, proved that red coloration is preferred during sexual selection due to its indication of higher predator defense. For further research into differences among diurnal and nocturnal beetles, I would increase my sample size and repeat the statistical analysis. Additionally, i n my data analysis and research, I was most fascinated by the concept of iridescence than any other phenotype among beetles. As a future project I would be interested in studying the functions of the different forms of iridescence in beetles Acknowled gements I would like to thank advisor Federico Chinchilla and peer reviewer Chris Cosma for their constructive criticisms on improving the deliverance of my study. Thank you to all my peers and instructors in the EAP program for their encouragement and re assurance throughout the process, especially those who helped me hunt for beetles. Finally, a grand gratitude goes out to my advisor Emilia Triana for her guidance, patience hours of dedication, her beautiful personal Coleoptera identification notes and for initially introducing me to the exciting world of arthropods. Without her, this project may have been instead a biod iversity comparison of mammals. I was told to find a mentor and I have found one in Emilia Triana.
Diurnal and nocturnal c oleopterans in the Monteverde z one Kim 10 LITERATURE CITED Bezzerides, A. L., McGraw, K. J., Parker, R. S., & Husseini, J. (2007). Elytra color as a signal of chemical defense in the Asian ladybird beetle Harmonia axyridis. Behavioral Ecology and Sociobiology 61 (9), 1401 1408. Crowson, R. A. (2013). The biology of the Col eoptera Academic Press. Daly, H. V., Doyen, J. T., & Ehrlich, P. R. (1978). Introduction to insect biology and diversity. McGraw Hill Book Company. Huffaker, C.B. and A.P. Gutierrez. (1999). Ecological entomology. John Wiley and Sons Inc. Jones, F. M. (1932). Insect coloration and the relative acceptability of insects to birds. Transactions of the Royal Entomological Society of London 80 (2), 345 371. Mason, C. W. (1927). Structural colors in insects. III. The Journal of Physical Chemistr y 31 (12), 1856 1872. Nadkarni, N., Wheelwright, N.T., (2000). Monteverde: Ecology and Conservation of a Tropical Cloud Forest Oxford UP. Parker, A. R. (1998). The diversity and implications of animal structural colours. Journal of Experimental Biology 201 (16), 2343 2347. Rosenthal, M (2006). Nocturnal vs. Diurnal Insect Diversity Within the Tropical Montane Forest Canopy. JYI The Undergraduate Research Journal Journal of Young Investigators. Seago, A. E., Brady, P., Vigneron, J. P., & Schultz, T. D. (2009). Gold bugs and beyond: a review of iridescence and structural colour mechanisms in beetles (Coleoptera). Journal of the Royal Society Interface 6 (Suppl 2), S165 S184. Thry, M., Pincebourde, S., & Feer F. (2008). Dusk light environment optimizes visual perception of conspecifics in crepuscular horned beetle. Behavioral Ecology, 19(3), 627 634. White, R. E. (1983). A field guide to the beetles of North America (2nd ed.). Boston, NY: Houghton Mifflin