Epicephala moths appear to use diapause at multiple stages in their lifecycle in order to deal with variable flowering phenology (Fig. 6). The close association between the abundances of fruits and moths suggested that moths may diapause as eggs or young larvae within pollinated flowers, appearing as adults as fruits mature. We confirmed this by showing that many overwintering flowers contained pollen and evidence of diapausing pollinators. Our observations suggest egg diapause within dormant flowers serves a critical function of physically linking the pollinator lifecycle to host plant flowering phenology, ensuring that pollinator emergence coincides with flowering. In addition, our study of adult eclosion times also indicate that 12% of the autumn generation of Epicephala enter a pre-pupal diapause, emerging up to one year later. As such, Epicephala moths appear to use diapause at multiple stages in their lifecycle in order to track host plant phenology and mitigate against environmental unpredictability.
Host plant phenology
Inter-annual rainfall can be highly variable within Australia, even when compared to environmentally similar areas elsewhere in the world [37, 53]. Indeed, many species of Australian plant are known to fruit and flower in response to variation in both rainfall and photoperiod [4, 5]. In B. oblongifolia, female flowers were present all year round, but their abundance varied widely and was best predicted by rainfall and photoperiod. In this way, B. oblongifolia seems typical of many Australian plants in that its reproductive phenology is adapted to environmental unpredictability. The effect of rainfall on a plant’s phenology is likely to be dependent on characteristics of the soil and surrounding landscape, as well as the frequency and intensity of previous rainfall events [6, 54, 55]. Given that soil moisture more accurately reflects the total water available to each individual plant, we believe that it is likely that soil moisture would also be a significant predictor of flowering and fruiting phenology if included in our model.
Interestingly, our winter flower surveys show that B. oblongifolia frequently retains pollinated flowers over winter. Our phenological model found that B. oblongifolia sets fruit in response to rainfall. Together, these two results suggest that B. oblongifolia can delay fruit maturation in pollinated flowers over the winter period and potentially also through the spring and summer depending on the prevailing environmental conditions. This likely explains why only female flowers are present during the winter, as well as how some fruits began to develop in the late winter of 2017, when both Epicephala moths and male flowers were absent (Fig. 3). Indeed, our own flower bagging experiments support this explanation, with many bagged (i.e., pollinator-excluded) branches developing fruits [56]. Retaining previously pollinated flowers that can develop to fruits in response to largely unpredictable rainfall is likely to make B. oblongifolia less vulnerable to environmental variability, thereby reducing the risk of fruits developing during periods of drought and potential reproductive failure.
The exact mechanism that allows B. oblongifolia to retain pollinated flowers and then develop them to fruits months later remains unclear. Pollen can remain viable for several hours to several months depending on the species and environmental conditions [57,58,59,60]. It may be the that the pollen on the overwintering flowers of B. oblongifolia have yet to germinate and does so shortly before fruit development. Currently, it is not known how long the pollen of B. oblongifolia can remain viable under natural environmental conditions. The fruits of several important crop plants are known to arrest their development in response to climatic conditions, with fruit growth initiating again following pollen fertilisation [61, 62]. As such, an alternative explanation could be that the retained pollinated flowers are already fertilised but remain in a developmental pause over winter until released by an environmental cue, such as lengthening photoperiod or increased soil moisture following rainfall. Further experimentation is required to answer these questions.
Pollinator phenology
Epicephala abundance at the Richmond field site in both the spring and summer of 2016–2017 and 2017–2018 occurred in two discrete peaks of high abundance, corresponding with similar peaks in fruit abundance (Fig. 3). Our analysis showed that the number of mature fruits was a significant predictor of Epicephala abundance. This is most likely because Epicephala moths emerge from mature fruits as larvae and then pupate to adults [14, 20]. The appearance of fruits is therefore an important predictor of adult moth abundance, as observed in some other leaf flower species [19,20,21]. Although we found no statistical evidence for a relationship between rainfall and moth abundance, there is an indirect relationship between the abundance of moths and rainfall via the abundance of fruits, which is rainfall dependent. Thus, rainfall and photoperiod are critical factors in the phenology of the B. oblongifolia host plant and, by extension, its Epicephala pollinators.
Consistent with our prediction, the abundance of both male flowers and Epicephala moths were best predicted by the mean minimum temperature 14–21 days prior to each plant and pollinator survey. Lepidoptera are often phenologically synchronised with their host plant’s growth stages through temperature [30,31,32,33,34]. As male flowers are a source of essential pollen and potentially nectar [19, 20], female Epicephala moths are likely to be under selection to synchronise their adult eclosion with the occurrence of male flowers. The fact that both male flowers and moths respond to the mean minimum temperature three weeks prior to each observation probably reflects the developmental time between the environmental trigger and appearance of mature fruits and moths. Indeed, the average time between the emergence of larvae from fruits and adult eclosion in our eclosion study was 3 to 4 weeks, strongly suggesting that temperature plays a critical role in synchronising moth and pollinator life histories.
The absence of adult moths during the winter months (June–August) and hottest summer months indicates that moths probably diapause during this stage in their lifecycle (Fig. 4). Furthermore, the close association between fruits and the abundance of adult moths suggests that these insects diapause as eggs, or possibly young larvae, within flowers that then develop to fruit. In line with our expectations, when we examined overwintering flowers, many showed scarring and boring damage consistent with Epicephala moths and their larvae [35, 36]. Although we did not survey flowers during the hottest summer months, we believe that it is very likely that moths also diapause as eggs in female flowers during this time. This is because all spring emerging larvae pupated directly to adulthood and did not enter a pre-pupal diapause. As such, spring emerging Epicephala presumably eclose, mate and lay diapausing eggs in female flowers that are present through the summer when adults are absent. Periods of egg dormancy are known in other Epicephala species [21] and many Australian Lepidoptera are also known to exhibit extended periods of egg diapause [27,28,29]. The larvae of some fig wasps are also known to overwinter within dormant fig flowers in temperate climates [22]. Our analysis of the phenological data has demonstrated that in B. oblongifolia, fruiting and flowering are phenologically synchronised with both fruits and new flowers appearing together. As such, egg or young larval diapause likely ensures that overwintering larvae develop and emerge at, or near, the time of flowering, creating a physical link between plant and pollinator lifecycles.
In moths, diapause can occur at both the egg and pre-pupal life history stages. Our study of the interval between larval emergence and eclosion found that 12% of the Epicephala larvae that emerged in the autumn entered a pre-pupal diapause for periods of up to 48 weeks. These long diapausing Epicephala included both species known to pollinate B. oblongifolia [35]. The majority of moths that emerged in the autumn pupated directly to adulthood, eclosing 3 to 4 weeks after leaving the fruits. Spring emerging Epicephala were not observed to enter an extended pre-pupal diapause, but this may reflect the lower sample size (n = 13). The varying lengths of pre-pupal diapause documented here may constitute a bet-hedging or risk-spreading strategy [63]. Bet-hedging strategies involve a loss of individual fitness to reduce variance in fitness over time, thereby increasing the long term (geometric) mean fitness of the genotype or lineage [64, 65]. Such strategies are adaptive in unpredictable environments, where variance in fitness between generations is high. In our example, moths that pupate directly to adults after emerging from fruits late in the growing season may experience shortages of flowers. A bet-hedging genotype that produces multiple phenotypes (e.g., differing diapause durations) may thereby achieve greater geometric fitness over time by reducing variance in fitness between flowering seasons. Bet-hedging strategies are commonly inferred in insects, but rarely shown definitively [63]. This is because such strategies must be correctly distinguished from genotypic polymorphism within populations [63]. Furthermore, demonstrating that bet-hedging strategies occur requires demonstrating greater geometric fitness in bet-hedging genotypes compared to none bet-hedging genotypes. Alternatively, it is also possible to demonstrate bet-hedging by showing variation in the trait in question (e.g., diapause duration) in relation to environmental uncertainty. Bet-hedging is, therefore, challenging to demonstrate experimentally. Regardless, our work is the first to suggest the possibility of a bet-hedging strategy in a nursery pollination mutualism.
Implications for the mutualism
Since they represent independent evolutionary origins of pollination mutualism, it is interesting to compare the phenology of plants and pollinators in the fig, Yucca, and leaf flower mutualisms. Based on available data, it would seem that, at the population level, the flowers of both fig trees and leaf flowers are generally present throughout the year [18,19,20,21, 24, 25]. However, as this study and others have demonstrated [21], flowers may be present but dormant for part of that time. In at least one temperate fig species, overwintering flowers act as refuges for fig wasp pollinators through the winter months [22]. In B. oblongifolia, and possibly other leaf flower plants, the majority of plants bear female flowers year-round, with low numbers also present during the winter. Year round flowering may thereby promote stable populations of Epicephala moths by providing a refuge for pollinators during periods when plants are not growing fruits or new flowers. This could explain why some B. oblongifolia plants maintained female flowers during periods of obvious drought stress in the unusually dry winter of 2017. Indeed, our observation that these pollinated and pollinator-containing flowers were maintained at the expense of leaf tissues suggests a high fitness value to the host plants. In the Yucca-yucca moth OPM, pollinators diapause as pre-pupal larvae in the soil around their host plants and may wait several years between the appearance of flowers [11]. The ability of yucca moths to diapause for multiple years between flowering events probably also promotes the stability of pollinator populations. Our study has found that both year-round flower provision and pre-pupal diapause occur in at least some leaf flower plants, showing similarities with both the Yucca and fig mutualisms.
Egg diapause in Epicephala may have other important implications for the mutualism. Overwintering larvae or eggs are likely to suffer moderate levels of mortality during diapause [66, 67]. This increased mortality may benefit the plant by reducing the number of seeds that are consumed by pollinators. Overwintering mortality could help to explain the large proportion of B. oblongifolia fruits (10–30%) that do not contain Epicephala larvae [36]. Fruits that do not contain pollinators are generally more frequent in crops collected in the spring and contribute a high proportion of the intact seeds produced across B. oblongifolia populations [36]. As such, egg diapause mortality may be an important factor in reducing seed destruction by pollinating seed herbivores, thereby helping to maintain the mutualism in the face of competing interests between mutualists.