How Often Do Males and Females Mate, and What Factors Affect the Timing of Mating?
(continued)

Abstract  |  Introduction  |  Methods & Results |  Discussion   |  Acknowledgments  |  References   |  Karen's Research Questions

Methods and Results

General Methods

For all mating experiments, we used offspring of wild-captured adults that were reared in captivity. Adults were marked with unique numbers the day after they eclosed. We fed them a 20-25% honey water solution daily, and kept them in outdoor net cages (2m x 2m x 2m) during the experiments. Butterfly densities were 15-25 individuals of each sex per cage, with 1:1 sex ratios in all experiments. Fresh milkweed plants were provided daily for oviposition.

We checked for mating pairs at two hours intervals throughout the day and recorded all matings. This frequency ensured that we observed all matings because monarchs begin mating in mid- to late afternoon and remain in copula for several hours, rarely separating before nightfall (Oberhauser 1989).

Age at first mating and mating frequency

In 1994, we kept groups of males and females together in cages throughout their lives, recording every mating. We used four cages, two that started with one day old females combined with five or six day old males, and two that started with one day old males combined with five or six day old females. All butterflies were kept in the same cages until they died. This allowed us to determine the age at which individuals of each sex would start mating; using older individuals of the other sex ensured the presence of mature individuals with which they could mate. Total sample sizes were 75 individuals of each sex.

We measured the mating frequency of females in 1986 differently. In this experiment, we kept 38 females in cages with males throughout their lives, but replaced males every day, so that the cages only contained males that were either unmated or had not mated within four days, and would thus transfer large spermatophores (see What factors affect the size and composition of monarch spermatophores?).

Figure 1 summarizes the age of first mating. The percentages of individuals that mated for the first time at different ages and the cumulative percent that mated on or before each age are shown as a function of age. Individuals that never mated are not included in the figure. Even when kept with older, sexually mature females, no males mated before the age of three days, and most of them first mated at ages five, six or seven days (figure 1a). The age at first mating was spread more evenly from one to nine days for females (figure 1b), with most females mating by four days of age when confined with older males. 

Figure 1: The age at which male and female monarchs first mated. Both the proportion of individuals that mated for the first time at each age, and the cumulative proportion that had mated by each age are shown.

In 1994, when the same males and females were kept together throughout their lives, males mated from zero to 17 times, with a mean of six matings, and a median of five (figure 2). Nine males (12%) never mated. Females mated from zero to 15 times, also with a mean of six matings and a median of five (figure 2). The distribution of mate number for males is much more skewed than for females, with more males falling into the low categories. The distribution for females closely approximates a normal distribution, with most females mating intermediate numbers of times. Only three females (4%) never mated.

Figure 2: Frequency distribution of the total number of matings for males and females kept in mating cages throughout their lives. The same individuals stayed in the cages for the entire experiment, so females received a variety of spermatophore sizes.

In 1988, when females were only exposed to males that transferred large spermatophores, the distribution of mating frequencies is similar in shape to that of 1994, but mate numbers ranged from one to six, with a mean of 3.5 (figure 3). Four of the females in this experiment, both of those that mated six times and two that mated four times, were killed when their bursa copulatrix (see Female anatomy) became so full that it ruptured. In each of these cases, females had mated at least three times within seven days (one mated four times in five days). When I dissected these females, the last spermatophores were not in the bursa copulatrix, and the sperm in them had not been transferred to the spermatheca.

Figure 3: Frequency distribution of the total number of matings for females kept in mating cages with males that would only transfer large spermatophores (unmated males, or males that had not mated for four or mare days).

Effects of Spermatophore Size on Intermating Intervals and Mating Probability

In females. We measured the time between the first and second matings of 70 females that received either large or small spermatophores in the first mating. All females mated for the first time at age six or seven days with either unmated males or males that had mated one to two days previously (spermatophore masses of approximately 30-37 mg and 7-15 mg respectively, Oberhauser 1988). Females were then kept in mating cages with males until they remated. (See Oberhauser 1989 for more details.)

Figure 4 shows the cumulative proportion of females that had remated as a function of time in the intermating interval study. Females mated to males that transferred large spermatophores waited longer to remate than those that received small spermatophores. 78% of those that received small spermatophores had remated three days after mating, while it took eight days for the same proportion of females that received large spermatophores to remate. The mean times to remating for females that received large or small spermatophores were 4.6 and 3.0 days, respectively.

Figure 4:  The cumulative proportion of females receiving small and large spermatophores that had remated each day after their initial mating. Females that received small spermatophores remated sooner.

In males. We conducted two experiments to determine whether males delay remating until they can produce larger spermatophores. First, we compared the likelihood that males with different mating histories would mate when exposed to females. Unmated males that were six or more days old, and males that had mated one to eight days previously were put into mating cages with six to ten day old unmated females. Since weather affects mating probability, we used males with many different mating histories simultaneously to even out weather effects.

The first experiment utilized unmated females who would presumably have a high propensity to mate. In the second experiment, we compared the mating likelihoods of males with different histories when they were kept with either mated females, who are less motivated to mate (see below), or unmated females who should be more willing to mate. On day 1 of this experiment, we released 40 unmated males and females into mating cages. Mated individuals from day 1 (30 of each sex) and 30 unmated individuals of each sex were assigned to four treatments on day 3 of the experiment: 15 mated males with 15 mated females, 15 mated males with 15 unmated females, 15 unmated males with 15 mated females, and 15 unmated males with 15 unmated females. None of the butterflies that had not mated on day 1 were reused on day 3.

Figure 5 shows the mating propensities of males with different mating histories, when confined with unmated females. Recently-mated males, who transfer smaller spermatophores, are as just likely to mate as males that have waited longer between matings. In fact, the smallest proportion of matings was by previously unmated males (labeled 0* on the x-axis in figure 5), who transfer the largest spermatophores.

Figure 5:  The probably that males with various mating histories would mate when put into mating cages with females. The numbers above each bar refer to the number of males with each history that were tested. * = unmated males. Males that will transfer a larger spermatophore are not more likely to mate than recently-mated males that will transfer a small spermatophore.

The second experiment on male mating propensities tested the effects of both male and female mating history on male mating likelihood. The number of matings that occurred in each of the treatments is shown in table 1. We used a loglinear model to test these data. The best model is one that includes an interaction between female history and the likelihood of mating, and none between male history and mating likelihood (G² = 0.85, df = 2, P = 0.659; the loglinear test compares models to find the one that best fits a data set, so a high P value corresponds to the best model). This means that females that had mated two days ago were less likely to mate, but that male mating history had no effect on mating likelihood.

Table 1. The number of pairs that mated in treatments with males and females of different mating histories. The total possible number of pairs in each treatment was 15.

Discussion

Age at first mating

The age at first mating for females confined with sexually mature males was spread relatively evenly over nine days, with almost half of the females mating by the age of three days (figure 1b). This is before they have mature eggs ready to fertilize, and earlier than we found in another study (Oberhauser & Hampton 1995, Does mating cause eggs to mature?). This result could be due to exceptionally favorable weather conditions during the experiment that could have increased mating probabilities. While we did not observe the initiation of all matings, we suspect that many of the early matings were forced by males (see How is monarch mating behavior different from that in other butterflies?). Aerial takedowns by male monarchs can result in forced copulation (Pliske 1975, Rothschild 1978, Oberhauser 1989, Van Hook 1993, Frey et al. 1998), even though females can usually evade unwanted mates (Oberhauser 1989, Frey et al. 1998, How is monarch mating behavior different from that in other butterflies?). While females could benefit from receiving male-derived nutrients at any time, there might be a cost to mating too early. Spermatophores can represent 5-10% of adult mass (Oberhauser 1988, 1992, Spermatophores; Svärd and Wiklund 1989), and degrade over a period of several days after mating (Oberhauser 1992). This means that mated females have an increased flight load that could make flying energetically more expensive, or decrease their ability to avoid predation. Other potentially relevant costs include the time spent mating, increased predation during mating itself, or sexually transmitted disease. It is possible that early-mating females gained some benefit from mating, but we think the fact that over half of the females did not mate before the age of four days, despite being in a cage with sexually mature males, suggests that early mating is not usually beneficial. Females that delay mating until they are ready to reproduce avoid costs associated with early mating while still obtaining nutrients to augment larval reserves early in the process of egg maturation.

The more compressed time interval over which males first mate (figure 1a) suggests that most males are not ready to mate until they are five days old. Only two out of 75 males mated before this age, but over half had mated by age seven days. This suggests that males are not physiologically ready to mate until they are about a week old. During their first few days of life, they accumulate accessory gland material to use in spermatophores (see What factors affect the size and composition of monarch spermatophores?), and it is possible that sperm maturation is not complete until a few days after they eclose.

The fact that most individuals of both sexes do not mate immediately after they eclose means that there is potential for movement away from the natal area before reproduction. This could help to prevent genetic differentiation between monarch populations in different locations (Eanes and Koehn 1978). It may be especially important as monarchs are migrating south in the spring, since monarchs can move relatively long distances before they begin to spend time mating and searching for oviposition sites.

Effects of Spermatophore Size on Female Monarch Intermating Interval

When male monarchs transferred larger spermatophores, females waited an average of almost two days longer to remate (figure 4). In monarchs, the last male that has mated with a female fertilizes most subsequent eggs (see Whose sperm fertilize the female’s eggs if she mates more than once?). Thus, a male will fertilize more eggs from a given mate if she does not mate soon after he mates with her. Since females often lay up to 100 eggs per day (see What factors affect the number of eggs that females lay?), this can represent an important difference in the value of a mating to a male.

Effects of Spermatophore Size on Male Mating Interval

I argued above that males benefit by producing a large spermatophore, since this increases the time that their mates will wait before remating. However, males in two experiments were just as likely to mate when they would only transfer a small spermatophore. This seems counter-productive, since they would transfer a larger spermatophore if they delayed mating for a few days. An explanation of this strategy can be sought using a cost-benefit analysis. If a recently mated male encounters a female, he can either attempt to mate with her, or wait until he is able to contribute a larger spermatophore to a future mate. Mating would deplete the small amount of accessory gland material that is available, and may not result in many offspring if the female remates soon. Waiting could increase the value of a future mating, since the future mate will probably wait longer to remate. However, giving up an opportunity to mate would be a costly strategy if the male is unlikely to find another mate. It may be that the chances of obtaining matings are low enough that it is always best for a male to mate whenever he can.

Monarch Lifetime Mating Frequencie

The shape of the frequency distributions of the number of lifetime matings for male and female monarchs are very different. Males have a skewed distribution with high variance, while females have a distribution that is very similar to a normal curve, with the number of matings for most females falling in the middle of the curve (figure 2). The se different shapes are typical of many animals (see Bateman 1948). The lower variation in female mate numbers suggests that most females probably mate close to the number of times that optimizes the number of offspring that they produce, although the ability of male monarchs to force unwilling females to mate could increase female mating frequency to a number higher than is optimal. The variation in male mating success suggests that some males have very low reproductive success (in fact 9% of the males in this study never mated), and a few have very high success (two males mated 17 times). This means that there is potential for strong sexual selection on male monarchs—selection that favors traits that make males more likely to mate with many females.

Acknowledgements

These experiments were conducted over several years, and many people helped with them. I especially thank C. Jessup, D. Alstad, W. Herman, O.R. Taylor, R. Rutowski, C. Boggs, P. Oberhauser, S. Oberhauser, B. Sharp, P. Van Meter, C. Wiklund, S. Stai, L. Goehring, D. Frey, D. Cansler, A. Feitl and P. Abrams for their help with the experiments or in data analysis and presentation. Financial support was provided by the NSF (BSR 8805884 and DEB 9220829), the University of Minnesota Graduate School, and the Dayton and Wilkie Funds for the Study of Natural History, administered by the Bell Museum of Natural History at the University of Minnesota.

return to top of page

References

Bateman AJ (1948) Intra-sexual selection in Drosophila. Heredity 2:349-368

Boggs CL (1981) Selection pressures affecting male nutrient investment at mating in heliconiine butterflies. Evolution 35: 931-940

Boggs CL (1990) Effects of male-donated nutrients on female fitness in insects. Amer Nat 136:598-617

Boggs CL, Gilbert LE (1979) Male contribution to egg production in butterflies: Evidence for transfer of nutrients at mating. Science 206: 83-84

Boggs CL, Watt WW (1981) Population structure of Pierid butterflies. I. Genetic and physiological investment in offspring by male Colias. Oecologia 50: 320-324

Eanes WF, Koehn RK (1978) An analysis of genetic structure in the monarch butterfly, Danaus plexippus L. Evolution 32:784-797

Frey, D., K.L.H. Leong, E. Peffer, R.K. Smidt, K. Oberhauser. (1998) Mate pairing patterns of monarch butterflies at a California overwintering site. J. Lep. Soc. 52: 84-97.

Greenfield MD (1982) The question of paternal investment in Lepidoptera: male-contributed proteins in Plodia interpunctella. Int J Inv Repro 5: 323-331

Oberhauser KS (1988) Male monarch butterfly spermatophore mass and mating strategies. Anim Behav 36: 1384-1388

Oberhauser KS (1989) Effects of spermatophores on male and female monarch butterfly reproductive success. Behav Ecol Sociobiol 25:237-246

Oberhauser KS (1992) Rate of ejaculate breakdown and intermating intervals in monarch butterflies. Behav. Ecol. Sociobiol. 31:367-373.

Oberhauser KS, R. Hampton (1995) Relationship between mating and oogenesis in monarch butterflies. J. Ins. Behav. 8:701-713.

Oberhauser, K. S. 1997. Fecundity and egg mass of monarch butterflies: effects of age, female size and mating history. Functional Ecology 11(2): 166-175.

Pliske TE (1975) Courtship behavior of the monarch butterfly, Danaus plexippus L. Ann Ent Soc Amer 68:143-151

Rothschild M (1978) Hell's angels. Antenna. 2, 38-39.

Rutowski RL, Gilchrist GW, Terkanian B (1987) Female butterflies mated with recently mated males show reduced reproductive output. Behav Ecol Sociobiol 20: 319-322

Rutowski RL, Long CE, Marshall, LD, Vetter RS (1981) Courtship solicitation by Colias females. Amer Midl Natur 105:334-340

Rutowski RL, Newton M, Schaefer J (1983) Interspecific variation in the size of the nutrient investment made by male butterflies during copulation. Evolution 37: 708-713

Svärd L, Wiklund C (1989) Size and production rate of ejaculates in relation to monandry/polyandry in butterflies. Behav Ecol Sociobiol 24:395-402

Wiklund C, Kaitala A, Lindfors V, Abenius J (1993) Polyandry and its effect on female reproduction in the green-veined butterfly (Pieris napi) Behav Ecol Sociobiol 33: 25-43

Watanabe M (1988) Multiple matings increase the fecundity of the yellow swallowtail butterfly, Papilio xuthus L., in summer generations. J Ins Behav 1:17-30

Van Hook, T. 1993. Non-random mating in monarch butterflies overwintering in Mexico. pp 49-60 in S.B. Malcolm and M.P. Zalucki, eds. Biology and conservation of the monarch butterfly. Natural History Museum of Los Angeles County, Science Series 38.

Return to top  |  Karen's Research Questions  |  Reproduction Home Page  |  Research Topics  |  Site Overview