We observed mating behavior in a South Indian population of M. leda in captivity, and measured mating duration and spermatophore size. We found that these butterflies have brief periods of mating behavior around sunrise and sunset, and that 42% of evening matings lasted through most of the night. Female M. leda are only moderately polyandrous and appear to exercise mate choice.
Most matings were initiated within a 15-minute time frame during both morning and evening observations. In contrast, Kemp (2002) did not report mating activity in the morning in Australia. Therefore, there may be differences among populations in the occurrence or relative frequency of morning and evening matings. Early morning mating behavior may be rare among butterflies (Freitas et al. 1997). Nevertheless, owl butterflies (Caligo idomenaeus) also show mating activity at dawn for only about 15 min (Freitas et al. 1997), and similar behavior might occur in other species of evening brown. We observed that M. leda butterflies can shiver in the morning, which is similar to owl butterflies (Srygley 1994), and hitherto unknown in this species. Shivering may allow butterflies to warm up and be active when the ambient temperature is too low for flight activity (Masters et al. 1988; Srygley 1994). Since shivering is energetically costly (Srygley 1994), females may be selected to prefer males that are able to compete when the ambient temperature is low. In addition, there may be selection for females to be receptive only during a short time-frame to promote competition among males. The timing and brevity of mating periods may also be selected by predators (suggested by Freitas et al. 1997) or to serve to avoid reproductive interference from other species that mate at different times of the day (suggested by DeVries et al. 2008).
The durations of morning matings were within the normal range recorded for satyrine butterflies (Molleman et al. 2004), but the long nocturnal matings were unexpected. Since such long nocturnal matings did not lead to heavier spermatophores, it seems unlikely that material is transferred throughout the entire mating. Perhaps such long matings carry little predation risk during the night, while copulating is risky during the day. When in copula, mobility can be greatly constrained so that couples are more susceptible to predation than single individuals (Magnhagen 1991; Almbro and Kullberg 2009). Because predation pressure may be higher during the day compared to the night (Seifert et al. 2016), butterflies mating after sunset might experience relaxed predation pressure against long matings compared to those mating in the morning. Furthermore, these butterflies are not active during the night and thus nocturnal mating does not strain their activity budget. Mate guarding may be an unsatisfactory explanation in this case, because there is no mate competition during the night and females do not normally mate in quick succession (pilot study and our spermatophore counts). Nevertheless, perhaps long nocturnal matings do influence female behavior. Nocturnal mating has also been documented in monarch butterflies in their winter roosts, where coupling takes place during the day, but sperm transfer only starts at night, which probably is a form of mate guarding (Svärd and Wiklund 1988). From a mechanistic perspective, we hypothesize that butterflies follow their diel activity pattern and fall asleep while in copula, and uncouple as they wake up in the morning (Helfrich-Förster 2018). We suspect that M. leda butterflies often remain in copula during the night because there is neither cost nor benefit of them doing anything else.
Mating duration (of first mating) might be expected to correlate with spermatophore size because transfer of material costs time. Because there was no significant difference in spermatophore mass between morning and evening matings, we suppose that the full duration of the long nocturnal matings does not represent transfer of material. We therefore limited this analysis to observations of matings lasting less than 3 h, thus allowing only a sample size of 11. One data point showed a large spermatophore for a brief mating (1 h and 10 min), being about twice the mass of spermatophores of other matings that lasted less than two hours. Only if we exclude this outlier, do we find that longer matings resulted in larger spermatophores. Therefore, more data are needed to establish whether or not there is a correlation between mating duration (of the first mating) and spermatophore size in M. leda.
We found that males did transfer slightly smaller spermatophores during subsequent matings (when long matings were included), similar to many other butterflies (Dewsbury 1982; Oberhauser 1988, 1992; Svärd and Wiklund 1986). However, mating duration was not correlated with mating order (excluding nocturnal matings that exceeded three hours), while in butterflies subsequent matings of a male are often longer and sometimes shorter than the first mating (Svärd and Wiklund 1986; Bissondath and Wiklund 1996; Oberhauser 1988, 1992; Molleman et al. 2004). We found no significant relationships between seasonal form, body size, or mating interval and spermatophore size or mating duration, but note that our sample sizes were small.
Monandrous butterfly species tend to have smaller spermatophores than do polyandrous species (Svärd and Wiklund 1989). Since females of M. leda usually mate only once or twice, we may expect quite small spermatophores in this species. Indeed, the spermatophores we found were smaller than those of highly polyandrous species (Oberhauser 1989; Karlsson 1998). If females mate only once, males may also have limited chances to mate multiply, and may thus be selected to invest most of their resources in their first mating, impairing their ability to mate again (Wedell et al. 2002; Molleman et al. 2004). Nevertheless, M. leda males were able to mate multiply even within the same day. This may be expected because the nutrient investment in spermatophores may be small (suggested by low mass). In our experiments, females sometimes twirled with males before courtship, but there are too few observations from the wild to determine if this also occurs in nature. Such male-female twirling may indicate that females can use twirling as a means to assess male quality. We confirmed that interference among males can be part of the male mating strategy (one similar observation in Kemp 2002). Pliske (1975) also observed such interference in the monarch butterfly in captivity so that the density of butterflies had to be limited to 40 to observe courtship behaviors. Although interfering males did not succeed in mating with females directly, they were successful in breaking up the courtship of others, so that these females would remain receptive. More precise ethological observations could be obtained using fewer individuals in larger cages, clearly marked sexes (e.g. using males and females of different seasonal forms reciprocally), and night vision video recordings.
Our results suggest that in M. leda, males compete mainly through the territorial defense that was studied by Kemp (2002, 2003) and possibly courtship, and to a lesser extent through providing large spermatophores to females, or sperm competition. Females appear to exert some control over the mating system because receptive females are known to actively entice males by flying conspicuously near male territories (Kemp 2002), females may test males by engaging in twirling similar to male-male competition (this study), and refuse copulations by pointing their abdomens upwards (this study). Females may choose males based on male fighting ability during cool parts of the day, and perhaps based on wing-fluttering performance during courtship (which might fan male pheromones over the female). Since during courtship the male is directly behind the female, and courtship takes place during twilight, females are not likely to use visual cues when assessing potential mates.
In conclusion, we found that M. leda in South India (1) performs mating behavior for short periods of time around sunrise and sunset, possibly facilitated by shivering, (2) evening matings can last extraordinarily long without resulting in larger spermatophores, (3) males can mate multiply, even twice during the same day, (4) subsequent matings of a male tend to yield spermatophores of slightly lower mass than the first but are of similar duration, (5) males actively interfere in courtship of others, (6) receptive females may twirl with males and can refuse copulation, and (7) females usually mate once or twice during their life time.