Background on Sexual Selection

Transcription

1 HOW-TO-DO-IT Mate Choice IN SOLDIER BEETLES: Field & Laboratory Experiments that Demonstrate Sexual Selection in Action P ERRI K. EASON P ETER T. S HERMAN Although the theory of evolution is the foundation of modern biology, students too rarely have an opportunity to watch selection operate in natural populations of animals (Culp, 1999). This lack may be partially responsible for the unfortunate ignorance of many people regarding the significance of evolution in biology. Laboratory exercises that directly study evolutionary processes can help to increase understanding of the principles of evolution (Rutledge & Warden, 2000; Storey, 1997). In addition, many people are hostile to evolutionary theory, regarding it as opposing certain religious beliefs (Meadows, Doster & Jackson, 2000; Rutledge & Warden, 2000); by allowing students to see evolution in action, teachers can help to reduce students resistance to evolutionary theory and to generate discussions about what evolution means in biology (Lawson, 1999). PERRI K. EASON is Associate Professor in the Biology Department at the University of Louisville, Louisville, KY 40292; perri.eason@louisville.edu. PETER T. SHERMAN is Assistant Professor in the Biology Department at Transylvania University, Lexington, KY The exercise we describe here focuses on the process of sexual selection. In this exercise, students use observations they make in the field to develop hypotheses about sexual selection, and then they test their own hypotheses by collecting morphological measurements in the lab. Students may then run additional experiments testing their hypotheses in the lab, if desired. The high level of student involvement in choosing the focus of their investigation in this exercise enhances both the students interest in the material and their learning about the topic. In addition, the results they obtain allow them to test some of the basic tenets of evolutionary thought and thus provide a glimpse of the work that forms the foundation of modern biology. Background on Sexual Selection Darwin proposed the theory of sexual selection to explain the existence of secondary sexual characteristics, which were traits that could not be explained by his theory of natural selection. His idea was that some individuals within a population reproduce more successfully than others due to the 436 THE AMERICAN BIOLOGY TEACHER, VOLUME 65, NO. 6, AUGUST 2003

2 effects of intersexual mate choice or of intrasexual competition. In mate choice, members of one sex, usually the female, prefer to mate with males that have certain traits. For example, peahens may preferentially mate with peacocks that have exceptionally long tails. In populations in which such preferences are general, sexual selection can lead either to the evolution of striking morphological features, such as the peacock s tail, or to sexual size dimorphism. In intrasexual competition, members of one sex, usually the male, compete with one another for access to mates. Intrasexual competition can similarly result in the evolution of elaborate traits and larger body sizes, as these traits may be used in fighting by the competing sex. How Can Sexual Selection Be Studied in a Teaching Laboratory? One way that biologists test the theories of sexual selection is to observe which individuals are successful in obtaining mates and determine whether these individuals differ in some way from the individuals that are unsuccessful. Males that obtain mates tend in many species, for example, to be larger or more brightly colored than males that do not obtain mates. Such observed differences between successful and unsuccessful individuals do not provide definitive evidence of sexual selection, which ultimately will depend on the success of a mating, the number of progeny produced, and the proportion of those progeny that grow to adulthood and themselves successfully reproduce. However, in practice, if the individuals that are mating differ significantly in some trait from those that are not mating, then biologists generally assume that sexual selection has been occurring. It is possible, then, to use mating patterns to investigate patterns of sexual selection and thus evolution. In addition, biologists can identify the features that were significant in sexual selection by looking at the characteristics that distinguish successful and unsuccessful individuals. By looking for differences between successful and unsuccessful individuals, students can begin to see how evolution might work in practice, how certain traits might change over time, and how a trait might spread within a population. If, for example, in a particular species, males with mates are bigger than males without mates, sexual selection is acting to increase male size, and one would expect an increase in average male size over time, if no other factors are counteracting the effects of the sexual selection. In the laboratory exercise that we describe here, students first go into the field to observe and collect soldier beetles. Students make hypotheses about mate choice or male-male competition based on their observations in the field. They collect paired male and female beetles and unpaired males. The students then bring the beetles back to the lab, where they measure them and use these data to test their hypotheses. Students may then expand this work by performing laboratory experiments to test their ideas further, or to test new hypotheses that they develop. In order to examine mating patterns in a laboratory exercise, the study animal must have characteristics that make it amenable for student work. The species should be abundant, readily observable, and, if possible, of small body size, which makes handling simpler. In addition, it is helpful if the species is easily acquired or captured and is sexually dimorphic, so that sexing individuals is a simple matter. We have been using in our labs a species of soldier beetle that meets these requirements. The Study Animal For these investigations, we used a well-studied species of soldier beetle, Chauliognathus pennsylvanicus (for a color illustration of this species, see Borror & White, 1998). This particular species occurs over a wide range in the eastern United States and can be found in early to mid fall at very high densities on flowering goldenrod (Solidago sp.). Males and females are bright orange and have black spots on their elytra, the hard casings that cover their wings. Males and females are similar in appearance, but the sexes can be distinguished by differences in their abdomens: Females have fatter, more pointed abdomens, and ventrally they have black bands stretching across each abdominal segment, rather than the two discrete dark areas found on most abdominal segments in males (Figure 1). Males and females also differ from one another in some behaviors. For example, a male that has copulated with a female commonly spends some time guarding his mate. A mate-guarding male rides on the back of the female he is guarding, preventing her from copulating with other males and thus increasing his chances of fathering offspring. Students can readily observe this mate-guarding behavior in the field. Testing Hypotheses in the Field Using soldier beetles collected in the field, students can compare the attributes of males that were found mating with the attributes of males that were not paired with females. For example, students can test whether successful males, i.e. those that were with females, are larger than unpaired males, or whether they weigh more than unpaired males. Previous studies on soldier beetles have shown that one trait that appears to be significant in mate choice is the size of the dark spot on the elytra: The beetles in some populations show assortative mating, with females that have bigger spots tending to mate with males that have bigger spots, and females that have MATE CHOICE IN SOLDIER BEETLES 437

3 Figure 1. Female (left) and male (right) soldier beetles. Note the fatter and more pointed abdomen of the female and the difference in the distribution of black pigment on the underside of the abdomen in females and males. smaller spots mating with males with smaller spots (McLain 1982, 1985). Thus another appropriate hypothesis might relate to whether the sizes of the elytral spots are correlated in mating pairs of beetles. If desired, a variety of hypotheses can then be generated from the field observations and those hypotheses tested in laboratory or field experiments; possible hypotheses for testing in the laboratory are discussed later in this article. Collecting Beetles in the Field Although either the students or the teacher can collect the beetles in the field, we have found that students take great pleasure in being in the field and that their field observations of mating beetles enhance their appreciation of, and interest in, their later collection of data in the lab. Accordingly, if at all possible, we recommend that students be involved in collecting the beetles. To find the beetles in the field, the class should go to an old field in which flowering goldenrod is abundant and look on the flowering heads for the beetles, which are conspicuous and thus readily located. Working in pairs or groups of four, students should collect: 1. Males that are mating with or guarding a female and their associated females 2. Males that are not with a female. The beetles can be easily collected by snapping off small parts of the flowering heads on which beetles are located and placing each bunch of flowers and its beetle(s) in a vial. The goldenrod flowers will provide food and moisture for the beetles, and they also make a convenient substrate for the beetles to climb on. In general, each beetle should be placed separately in a small vial with a lid (inexpensive plastic vials can be ordered from BioQuip Products, Inc., Catalog # 8909, for 9 dram plastic tubes with snap on lids). However, copulating males and females should not be separated from one another because forcing them apart could damage their genitalia; accordingly, each copulating pair should be placed into the same vial for transport back to the lab. Upon arrival back at the lab, these individuals will be likely to have finished mating and can then be placed into separate vials. If a copulating pair separates on its own during collection, the two individuals can be placed in separate vials in the field. Each vial should be marked to indicate which category of beetle is placed in it (paired female, paired male, or unpaired male). A piece of matte finish cellophane tape can be placed on the lids of the vials, marked with pencil. Students should be careful to indicate pairs, i.e. if a female was captured with a male, they should note this on the vials into which they place these two individuals. For example, mated pairs could be indicated by allotting members of a pair the same number, e.g. Male #1 and Female #1. These notations will allow students to identify individuals for comparing the morphology of females and their mates. Each group should keep their vials in a separate bag to facilitate numbering the beetles and the relocation of specimens for measurement back at the lab. Following the lab, all beetles should be returned to the field in which they were caught, and released. Measuring Beetles in the Lab In order to measure the beetles sizes, it is necessary to slow the beetles by placing them in an ice chest or refrigerator. If the students are collecting beetles for use on the day after they are collected, the beetles should be kept in the vials in the refrigerator overnight. The soldier beetles warm back up rather quickly, so students should take out only one beetle at a time for measuring, 438 THE AMERICAN BIOLOGY TEACHER, VOLUME 65, NO. 6, AUGUST 2003

4 leaving the remaining beetles in the refrigerator. Once the beetles are chilled, there are two ways students can measure them. If no computers are available, students can simply measure the beetles by hand, using either a ruler divided into millimeters or calipers. If the laboratory is equipped with a computer and some means of putting images on the computer, students can instead use free, downloadable software for analyzing beetle sizes. We have found that the computers provide more accuracy and create more excitement about the project among the students. We describe in detail in the Appendix how to take the data using digitized images. Students measuring the beetles by hand should follow steps #2, #3, #9 - #11 and #13 in the Appendix under Measuring the Beetles Elytrum Lengths. For each beetle, students should measure the length of an elytrum, i.e., the distance from the line separating the thorax and abdomen to the caudal end of the elytrum (Figure 2). For consistency, the instructor should tell all students to take their measurements on one side of each beetle, i.e. they should measure either all left elytra or all right elytra. In more advanced classes, students may also want to measure the length of the spot on the elytra of the beetles; the same methods given in the Appendix for measuring elytrum lengths can be used for measuring the spots. If students develop hypotheses about the effects of weight differences on mating success, they should also weigh the beetles as described in the Appendix (steps #1 #4 under Weighing Beetles ). Analyzing Beetle Measurement Data After students have finished measuring and weighing their beetles, they should hand in their data sheets so that the results can be summarized. Several graphs can then be plotted to determine whether any trends are readily apparent in the data. Below we give sample questions that you can ask using the measurement data. 1. Is female mate choice likely to result in selection for particular male traits? Figure 2. The length of the elytrum (left arrow) is calculated by measuring from the midpoint of the anterior portion of the elytrum to the tip of the posterior part.the length of the elytral spot (right arrow) is calculated by measuring from the most anterior to the most posterior portions of the spot. Students often predict that larger males will be more likely to obtain matings with females. To assess whether males of a particular size are more likely to acquire mates than other males, students can compare the elytra lengths of mated and unmated males. The data generated by our students suggested that females preferred males that were slightly smaller than average size, and that females tended not to mate with the largest or the smallest males in the population (Figure 3). To examine the effects of weight, a similar graph can also be plotted substituting weight for elytra length. Similarly, to investigate whether the elytral spots affect female choice, students could compare the lengths of elytral spots in mated and unmated males. 2. Are females mating assortatively? To determine whether females are mating assortatively, e.g., pairing with males that are of a size similar to their own, students can construct a scatter plot that shows the elytra lengths for all mating pairs. Our data show the plotted points clustering around a vertical line, suggesting that females prefer average-sized males regardless of their own size (Figure 4). If females were mating assortatively, one would expect to see the plotted points clustering around a 45 degree diagonal line. Again, similar graphs can be plotted to compare the weights of mating pairs and the elytral spots of mating pairs. Performing Follow-up Experiments in the Laboratory Female soldier beetles mate more than once, allowing students to conduct mate choice experiments in the laboratory using the beetles collected in the field. We like to have our students plot the summary graphs discussed above and then use them to generate hypotheses that they would be interested in testing in lab. One hypothesis that our students commonly choose to test is that males that were paired with a female in the MATE CHOICE IN SOLDIER BEETLES 439

5 Figure 3. A comparison of wing (elytra) lengths of males that were collected from the field while mating with a female and unmated males. Number of Males Unmated Male Mated Male Males Wing Length (mm) Figure 4. A comparison of wing (elytra) lengths of females and the males that they were mating with when collected from the field. Females' Wing Length (mm) Males Wing Length (mm) field are more likely to mate with a female in the lab than are males that were not paired with a female in the field. This hypothesis suggests that either females consistently prefer a certain type of male, or a certain type of male tends to win in malemale competition over mates. Below we describe the procedure used for testing this hypothesis; these instructions include collecting observational data that allow students to determine whether female choice is occurring and whether malemale competition is influencing mating patterns. Following these instructions, we briefly discuss other hypotheses that might be tested using similar procedures. To determine whether previously successful male beetles are more likely to mate than males that had not been mating when they were collected, students should work in groups of four and follow the steps below: 1. Remove from the refrigerator: a) One male that was collected while paired with a female b) A similarly sized male that was solitary when he was collected c) A female. In order to avoid confusing their data, students should make certain that the female they choose is not the female that was previously mating with the male that they choose for their trial. 2. Mark the males by using a toothpick to place a tiny dot of acrylic paint on each male s thorax. The thorax is the section of the body that is below the head and above the elytra; it has a black rectangular square in this species. 440 THE AMERICAN BIOLOGY TEACHER, VOLUME 65, NO. 6, AUGUST 2003

6 These paint dots serve to identify the males; accordingly, students should use different colors for the two males so that they can be individually recognized. 3. Place the female and a goldenrod flowerhead in the center of a rectangular covered box approximately 20 cm x 30 cm. The box can be made out of a plastic storage container covered with screening, Plexiglas, or plastic wrap held in place with a large rubber band. Place the two males on opposite sides of the box at equal distances from the female. Record the measurements previously collected for each individual (e.g. elytrum length, elytral spots, weight) on the data sheet. 4. Observe any interactions and record the following data: How long it takes (in minutes and seconds) before mating occurs. Students should start timing when they put the insects into the box. One student in each group should perform a focal sample on the female to determine in what manner the mating occurs. A focal sample is a record of the behavior of one individual. Watch the female and record if she approaches a male, if a male approaches her, and what happens. Does she remain close to male or withdraw from him? Does the female simply mate with first male she encounters, or does it appear that she is actively choosing the male that she prefers? Another student should record any interactions that occur between males. Which male is dominant and able to displace the other? After mating occurs, does the male that is not mating try to interfere? How many times does he try to interfere? Is he successful in interrupting the mating? Students should observe the beetles for 10 minutes after mating begins to record any attempts at interference, or for a total of 15 minutes if no mating occurs. 5. After watching mating for 10 minutes, collect the individuals from the trial and put them back in the refrigerator in their original vials, which should be marked used to avoid accidental re-testing. 6. Start another trial following the same procedures. Typically, for a two-hour lab period we have each group of students run four trials, which leaves enough time to summarize and begin discussing results. To examine whether females prefer previously successful males, each group should report which males mated with the females in the lab trials, the previously mated males or the previously unmated males. Using data from all the groups, students can then perform a binomial test to determine whether previously mated males were preferred more often by females than would be predicted by chance. Students can also use their data to examine whether females are primarily responsible for mating patterns observed or whether male-male competition plays a role. If male-male competition plays a role, for example, one would expect that males might interact aggressively with one another, and males that won the interactions would tend to have characteristics similar to the males found mating in the field. Other Hypotheses To Test in the Lab Students may be interested in testing whether patterns observed in the field hold in laboratory conditions. For example, from our data they might choose to test whether average-sized males are more likely to achieve matings than longer or shorter males, or whether males of average weight have higher mating success than do heavy or light males. Students could also test whether females prefer males that have spots on their elytra that are similar in size to their own spots. To test this, each female to be tested should be placed in a test box with two males, one with elytra spots that are similar in size to those of the female and one with bigger or smaller elytra spots. Students could then combine their results to test whether females prefer to mate with males that are similar to themselves with regard to the elytral spots. Conclusions The laboratory exercises described above are highly flexible, providing multiple opportunities for students to choose the hypotheses on which they want to focus. Our students have greatly enjoyed this freedom, and we have found that by selecting their own hypotheses, they become more involved and interested in the experiments. In addition, no matter what result the students obtain, it is of interest in terms of evolution, and the data provide a snapshot of the process of evolutionary change that can serve as a springboard for discussion in the classroom. In our class, for example, we found stabilizing selection on male size and no assortative mating by size, but previous work on another population of soldier beetles found directional selection and assortative mating. Either of these results allows students to envision how sexual selection can operate to promote morphological change over time. We have found that our students have a greatly increased understanding of the processes of evolution when they have had the opportunity to see in the lab how selection might be operating in the field. MATE CHOICE IN SOLDIER BEETLES 441

7 References Borror, D.J. & White, R.E. (1998). A field guide to insects: America North of Mexico. Boston, MA: Houghton Mifflin Company. Culp, T. (1999). Demonstrating natural selection using magnetobacteria. The American Biology Teacher, 61, Lawson, A.E. (1999). A scientific approach to teaching about evolution & special creation. The American Biology Teacher, 61, McLain, D.K. (1982). Density dependent sexual selection and positive phenotypic assortative mating in natural populations of the soldier beetle, Chauliognathus pennsylvanicus. Evolution, 36, McLain, D.K. (1985). Clinal variation in morphology and assortative mating in the soldier beetle, Chauliognathus pennsylvanicus (Coleoptera: Cantharidae). Biological Journal of the Linnean Society, 25, Meadows, L., Doster, E. & Jackson, D.F. (2000). Managing the conflict between evolution and religion. The American Biology Teacher, 62, Rutledge, M.L. & Warden, M.A. (2000). Evolutionary theory, the nature of science & high school biology teachers: critical relationships. The American Biology Teacher, 62, Storey, R.D. (1997). A plea to college biology professors: It s time to move Darwin and his teammates from the bull pen to the starting lineup. The American Biology Teacher, 59, Appendix Using Computers & Image Analysis Software If the laboratory is suitably equipped, students can measure the beetles using a variety of software programs. To use these programs, you will need a computer that has a video input jack, a video camera with a video output jack, and a cable that can connect the two. If you are using a consumer video camera, you can attach it to a tripod and tilt the tripod head so that the camera is perpendicular to the surface on which you will be measuring beetles. Be aware that some consumer video cameras, once turned on, will only remain in stand-by mode for several minutes before automatically shutting off. This problem can be avoided by pressing the record button and videotaping the proceedings (the tape can be rewound and re-used as needed). An alternative method is to use a specialized video camera that can be attached with an adapter to the head of a stereo microscope. The software program that we use is called Image. This public domain program is available from the National Institute of Health (NIH) and can be downloaded from their web site ( nih-image/) along with an easy-to-follow manual that explains how to use the software. If you choose to use this program for the lab exercise described here, you should also download Plug-in Digitizer from the NIH web site. This program allows you to acquire images from a video camera and put them on a computer. The NIH Image program is designed for Macintosh computers; the NIH web site suggests alternative free software for PC computers that could also be used in this lab exercise, with slight modifications of the instructions given here. Calibrating NIH Image In order to measure the beetles, first the image analysis program must be calibrated so that a given length is known. The easiest way to perform this calibration is to place a ruler under the video camera and use that ruler to determine length. The video camera should be adjusted so that the field of view is between 4 and 8 cm, and the ruler should be placed on top of a Petri dish so that it will be at the same height at which the beetles will be measured (see the section below on measuring beetles elytrum lengths). If there are several measuring stations available to the students, the teacher may want to calibrate the measurements before class to save time. 442 THE AMERICAN BIOLOGY TEACHER, VOLUME 65, NO. 6, AUGUST 2003

8 Detailed instructions for calibration follow for those not familiar with using image analysis software: 1. Start NIH Image by double clicking on the Microscope icon in the NIH Image file. 2. Under File menu, choose Acquire and then highlight Plug-in Digitizer, which is to the right of Acquire. 3. Place the ruler on a Petri dish under the microscope. 4. Click OK and wait a moment. 5. Choose the dotted line tool from the tool menu. 6. Place the cross hairs on the screen at one end of the ruler and push down the mouse button there. Then, holding down the mouse button, drag the line to measure a distance of 30 mm. 7. Under Analyze menu, select Set scale. 8. First, enter the units that you measured (mm) and second, enter the number of those units that you measured on the ruler (30). 9. Click OK. Now the scale is set and the system is ready to measure beetles. 10.Under Analyze menu, select Show results. This opens up a results box that will show the results of all subsequent measurements that students make. Measuring the Beetles Elytrum Lengths In order to measure elytrum lengths using a computer, images of the beetles must first be acquired by the computer. Several different methods can be used to acquire the images. Below we give instructions for using a video camera to make measurements (steps #1 - #5 are the steps that acquire the image). As an alternative, one could take digital photographs and transfer those images to the computer. To measure the elytrum lengths: 1. Under File menu, choose Acquire and then, holding down the mouse button, highlight Plugin Digitizer, which is to the right of Acquire. 2. Remove one insect from the refrigerator or cooler and place it on foam in a Petri dish with its back facing upwards. (The foam should be cut into a disk that fills the Petri dish and should be 2 mm shorter than the height of the dish. The foam helps to keep the beetles horizontal and prevents them from crawling out of the field of view as they begin to warm up). 3. Put a glass cover on the beetle in the Petri dish to hold it flat. 4. Center the beetle s elytra in the field of view on the computer screen. 5. Click OK and wait a moment. 6. Place the cross hairs on the screen at the base of the insect elytrum. Click the mouse and, holding down the mouse button, drag the line to the top of the insect elytrum and then release the button. 7. Under Analyze menu, select Measure. 8. Look in the results box on the lower right corner of the screen and record the length that you just measured on your data sheet. The length is in the units that you used to calibrate the measurements; mm are most appropriate. 9. Record this length on your data sheet. 10. Label the vial with a number, and record this number on your data sheet by the beetle s elytrum length. You need to keep track of which beetle is which so you can compare mated and unmated males and so that you can compare mated males and the females with which they are mated. In addition, you may want to investigate the weights of the beetles as well as their lengths, as we do in our labs (see below). 11. Put the beetle that you measured back into its vial and then return the vial to the refrigerator. 12. Click the Close Box in the upper left corner of the image box to remove the images on the screen (i.e. don t save them). 13.Start this process over with another beetle. Weighing Beetles We used electronic scales and gave our students the instructions below for weighing the beetles; the beetles weights are typically around g. 1. Remove a vial with a beetle in it from the refrigerator. 2. Put the vial containing the beetle on the scale and allow the scale to calculate the weight. 3. Then remove the beetle, place it in a different container to prevent it from escaping, and reweigh the empty vial. 4. To obtain the weight of the beetle, subtract the weight of the empty vial (Step 3) from the weight of the vial and beetle together (Step 2). MATE CHOICE IN SOLDIER BEETLES 443