Australian Journal of Zoology

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1 CSIRO PUBLISHING Australian Journal of Zoology Volume 48, 2000 CSIRO Australia 2000 A journal for the publication of the results of original scientific research in all branches of zoology, except the taxonomy of invertebrates All enquiries and manuscripts should be directed to Australian Journal of Zoology CSIRO PUBLISHING PO Box 1139 (150 Oxford St) Collingwood Telephone: Vic Facsimile: Australia david.morton@publish.csiro.au Published by CSIRO PUBLISHING for CSIRO Australia and the Australian Academy of Science

2 CSIRO 2000 Australian Journal of Zoology, 2000, 48, The basis of life-history plasticity in the tropical butterfly Hypolimnas bolina (L.) (Lepidoptera : Nymphalidae) Darrell J. Kemp School of Tropical Biology, James Cook University, PO Box 6811, Cairns, Qld 4870, Australia. Abstract The common eggfly, Hypolimnas bolina (L.) (Lepidoptera : Nymphalidae), is an adult-diapausing tropical and sub-tropical species that exhibits seasonal plasticity in adult body size. Here I investigated (a) whether size plasticity in this species is due solely to variations in rearing temperature, or whether photoperiod is also involved, and (b) whether rearing photoperiod affects the timing of ovarian development in adults. Individuals were cultured at temperatures ranging from 21 C to 30 C, and under daylengths of 11.5, 12.5 and 13.5 h in two separate experiments. Significant plasticity in juvenile developmental traits was detected in response to both variables, with cooler temperatures and shorter daylengths both leading to decreased developmental rates and increased size at maturity. Although juveniles grew more slowly with decreasing temperature, they grew faster with decreasing daylength. The timing of ovarian maturation was also related to rearing photoperiod; whereas long day adults (13.5-h photoperiod) had gravid ovaries after 10 days, the ovaries of short day adults (11.5-h photoperiod) were either regressed or nearly so. These preliminary results suggest that size variation observed in field populations of H. bolina may not be wholly accounted for by variations in developmental temperature and, furthermore, that photoperiod may be used by this tropical species as an environmental cue for the seasonal timing of reproduction. Introduction Most insect species exhibit some degree of variation in morphological and life-history characters such as size, colour, growth and developmental rates, and the timing of reproduction (e.g. Holloway 1993; Andersen 1993; Braby 1994; Gotthard et al. 1999). Although a sizable proportion of phenotypic variation can be attributed to genetic differences (e.g. Clarke and Sheppard 1975; Gordon and Smith 1988), examples of environmentally mediated variation are becoming increasingly common. In these cases, individual genotypes have the ability to produce alternative phenotypes depending on environmental conditions. This phenomenon, known as phenotypic plasticity, has become a major focus in the study of the evolution of phenotypic diversity in animals, as well as in plants (reviews in Schlichting 1986; Gotthard and Nylin 1995). Adaptive models predict that animals should evolve plasticity in order to maximise their fitness in seasonal environments, that is, environments where the optimum phenotype (for maximisation of reproductive success) varies seasonally (Gotthard and Nylin 1995; Nylin and Gotthard 1998). This hypothesis has been extended to explain the behavioural, physiological and/or morphological variation exhibited by insect species with seasonal diapausing generations (for examples see Hazel and West 1983; Harada and Numata 1993; Braby 1994; Yoshio and Ishii 1994). In these species, individuals use predictable seasonal cues, such as photoperiod, to synchronise their phenotype with the appropriate season (Danilevskii 1965; Tauber et al. 1986; Danks 1987). Temperate-zone butterflies, in particular, have been shown to use photoperiod as a basis for making decisions (Nylin 1994; Gotthard et al. 1999) about how fast to grow and develop, at what size to mature, and whether or not to enter a state of reproductive diapause /ZO X/00/010067

3 68 D. J. Kemp (Clarke and Platt 1969; Sims 1980; Hazel and West 1983; Pullin 1986; Nylin 1989; Yoshio and Ishii 1994; Nylin et al. 1995, 1996; Leimar 1996). Although reproductive diapause itself is accepted as a genuine adaptation to environmental seasonality (Tauber et al. 1986; Danks 1987), associated forms of life-history plasticity, such as variation in size at maturity, are less obviously adaptive (Gotthard and Nylin 1995). This is because environmental factors such as temperature may have direct physiological effects on the growth and development of poikilothermic organisms. For instance, a juvenile insect is expected to develop faster under higher temperatures and, in some cases, this can lead to smaller size at maturity (Atkinson 1994; Berrigan and Charnov 1994). Although some aspects of temperature responses could be adaptive, phenotypic variation arising in this particular instance might also represent an incidental result of developmental instability (Gotthard and Nylin 1995; Spitze and Sadler 1996; Nylin and Gotthard 1998). On the other hand, phenotypic variation on the basis of other environmental variables, such as photoperiod, are more likely to represent true seasonal adaptations (Danilevskii 1965; Tauber et al. 1986; Danks 1987; Nylin and Gotthard 1998). This makes it particularly important to identify and explore the underlying environmental basis of observed life-history variation in poikilothermic organisms, so that the likelihood of adaptive hypotheses can be properly evaluated. Information regarding reproductive diapause and allied forms of phenotypic plasticity in tropical insects is relatively sparse (Danks 1987). Although tropical insects may have the capacity for true reproductive diapause (Denlinger 1986; Jones and Rienks 1987; Braby 1995, and references therein), the environmental cues used to mediate this diapause remain largely unknown (Braby 1995). Seasonality in tropical habitats is driven primarily by variations in rainfall rather than temperature, and hence it is often less predictable than seasonality in temperate latitudes (Denlinger 1986; Jones and Rienks 1987). In a review of rainfall patterns for Townsville, Jones (1987) reported that the wet season may begin any time from October to March, and last for 1 6 months. Because rainfall determines seasonal adversity in the wet dry tropics (Jones 1987; Braby 1995 and references therein), annually predictable cues widely used by temperate-zone organisms, such as photoperiod, may be less reliable predictors of seasonal adversity in these areas. The relevance of this environmental variable as a seasonal cue for adaptive decision making (Gotthard et al. 1999) by butterflies at tropical latitudes is therefore worthy of investigation. Hypolimnas bolina (L.) is a tropical and sub-tropical nymphalid butterfly that exhibits phenotypic plasticity with respect to both life history and morphological traits. In north Queensland, adults of this species overwinter in sheltered gullies and creek lines from April to early September, and females taken from these sites have regressed ovaries and extensive fat bodies (Jones 1987), which is suggestive of a true reproductive diapause (Danks 1987). This breeding phenology, in relation to the annual variation in temperature and photoperiod at Cairns (16 53 S, E), is summarised in Fig. 1. At present the cues to the induction of diapause are not known, although individuals begin overwintering at a very similar time each year regardless of weather conditions (Kemp, unpublished data), which suggests that photoperiod may play a role (Jones and Rienks 1987). In addition to ovarian dormancy, diapausing individuals are significantly larger (as indicated by forewing length) than their directly reproducing wet-season counterparts (Kemp, unpublished), which indicates that this species also exhibits plasticity with respect to size at maturity. Here I investigate the environmental basis of life-history plasticity in this species by rearing individuals under conditions of varying temperature and photoperiod. Two sets of hypotheses are addressed. Firstly, since H. bolina enter overwintering sites in north Queensland in late March regardless of prevailing weather, it is possible that diapause induction is under photoperiodic control. This hypothesis predicts that individuals reared under short day conditions (less than approximately 12.5 hours daylight; Fig. 1) should show delayed ovarian development, relative to individuals reared under long day conditions. The second set of hypotheses deals with variation in body size. Since overwintering generations in this species are relatively large, size variation could represent an adaptive response to seasonality (that is,

4 Life-history plasticity in Hypolimnas bolina 69 Fig. 1. Annual variation in temperature (dotted line) and photoperiod (solid line), in relation to the period of year in which adult H. bolina overwinter (shaded area) at Cairns (16 53 S, E). Temperature data is from Bureau of Meteorology records dating from 1941 to 1996, and daylength was calculated using a solar angle calculator (Seattle Energy Works). functionally linked with reproductive diapause). Large size would afford an overwintering individual greater capacity for storage of water and nutrients required for prolonged survival (Chaplin and Wells 1982; Danks 1987; Ohgushi 1996). This adaptive hypothesis makes the prediction that if photoperiod-mediated size plasticity occurs, it should proceed in the direction of larger adults under shorter-day conditions. Alternatively, the non-adaptive explanation for size variation is that it stems entirely from the physiological effect of temperature on the developmental and growth rates of juveniles (Atkinson 1994). This incidental effects hypothesis predicts that variation in developmental traits should proceed solely on the basis of developmental temperature alone. Methods Rearing protocols Two sets of rearings were conducted. In both cases, eggs were obtained by placing field-caught (Townsville, S, E) females individually into sealed clear plastic buckets (height 175 mm, radius 110 mm) containing fresh growth of S. nodiflora, their favoured oviposition plant (Kemp 1998). Individual buckets were placed outside in dappled sunlight for 2 3 h, after which all eggs were removed and placed in plastic Petri dishes lined with moistened absorbent paper. The pool of eggs derived from all females were distributed randomly amongst the treatments of each rearing set. In the first rearing set (conducted October December 1998), eggs obtained from six females were divided amongst four constant-temperature treatments: 21, 24, 27 and 30 ± 0.5 C. All treatments were set at a 13 : 11 L : D photoperiod cycle. Eggs were checked hourly on the day of hatching (eggs turn dark one day prior to hatching: Clarke and Sheppard 1975). Newly hatched larvae were transported in groups of to sealed rectangular plastic containers (approximately mm) lined with moist absorbent paper and containing fresh cuttings of Asystasia gangetica (Acanthaceae). Humidity within the individual rearing containers was maintained at a high level to prevent moisture loss from foodplant cuttings. Larvae were reared in groups of until their fourth instar, when they were reared in groups of 10 to a container and then in groups of 4 in their last (fifth) instar. Containers were cleaned daily (washed with 1% sodium hypochlorite then dried) and a new sheet of absorbent paper added each day. Cuttings of the foodplant (A. gangetica) were collected from a shaded streambank area in Cairns each day, soaked in 1% bleach for 10

5 70 D. J. Kemp min, then double rinsed before being fed to larvae. Larvae and pupae were checked once each day for pupation and emergence, respectively, with individuals being weighed to the nearest g on the day following pupation. After emergence, adults were sexed, and the length of their forewing was measured (from apex to insertion) to the nearest 0.5 mm using plastic callipers. In the second rearing set (conducted April 1998), animals were cultured using the methodology described above at a constant 27 ± 0.5 C and three photoperiod treatments: 11.5, 12.5 and 13.5 light hours in a 24-hour cycle. The mean temperature inside each cabinet did not vary depending on whether the lights were on (comparison of 10 temperature measurements at 30-min intervals, in both dark and light, t 18 < 0.193, P > 0.75 for all three cabinets). Eggs were derived from five females, and owing to shortages in A. gangetica, individuals in all treatments were cultured on S. nodiflora, which was collected on campus at James Cook University, Cairns. Rearing protocols were as above except neither foodplant cuttings nor plastic rearing containers were washed with sodium hypochlorite. Pupae were weighed to the nearest g at 24 h after pupation; however, each pupa could not be sexed individually upon emergence. Freshly emerged adult females in the photoperiod trial were placed inside a flight cage (dimensions m) on the James Cook University campus for 10 days, after which they were dissected to determine their reproductive status. A calibrated eyepiece micrometer was used to measure the length of oocytes to the nearest 0.1 mm (using 40 magnification). Ovarian development was assessed on a scale of 1 4 as follows: 1 = eggs unexpanded, no eggs discernible within the ovarioles (magnification 40); 2 = most mature eggs partially expanded, white, mm in length; 3 = most mature eggs green, mm in length; 4 = most mature eggs chorionated, >0.25 mm in length. The amount of fat body was assessed on a 4-point scale (none, scarce, moderate, extensive) as per Braby (1995). The forewing length of dissected females was measured to the nearest 0.5 mm. Statistical procedures The effect of rearing variables on juvenile development was evaluated with regard to three life-history traits: developmental rate, larval growth rate, and pupal weight. Developmental rate was defined as the proportion of development completed in 1 day, and calculated as the reciprocal of total developmental time, in days, from egg to adult. Larval and pupal developmental rates, where calculated, used only the proportion of developmental time in each respective life stage. Since butterfly larvae usually grow exponentially (Gotthard et al. 1999), growth rate was calculated by the following formula: log (r) = log (w)/d where r is the daily growth rate, w is the pupal weight, and d is the number of days from egg to pupa (Nylin et al. 1989; Nylin 1992). Analysis of variance (ANOVA) was used to test for differences in life-history traits between individuals of each treatment and, in the case of temperature rearings, between the two sexes. Sex-based differences in temperature effects were given by the interaction term between temperature and sex in a 2-way factorial design. Prior to conducting all tests, dependent variables were screened for normality using the Kolmogorov Smirnov goodness of fit, and Levene s test was used to confirm homoscedasticity. Factorial analyses were conducted using Type III sums of squares in order to account for unbalanced designs (Shaw and Mitchell-Olds 1993). Results Temperature effects on juvenile development All studied life-history traits (growth and developmental rates, adult size) varied significantly across the individual temperature treatments and between the sexes (Table 1). On average, butterflies reared under cooler temperatures grew more slowly, developed more slowly, and matured at a larger size than those grown under warmer temperatures (Fig. 2). With variation in the weight of different pupae accounted for (entering pupal weight as a covariate in an ANCOVA), pupae also developed more slowly under cooler temperatures. Compared with males, females developed more slowly as larvae and pupae (with pupal weight variation accounted for using ANCOVA), and matured at a larger size (Fig. 2). Rates of mortality were 5 8% (Table 2), and did not appear to relate to rearing temperature. The sexes differed in their response to temperature variation, at least in terms of larval and pupal developmental rates (indicated by the significant interaction terms between sex and

6 Life-history plasticity in Hypolimnas bolina 71 Table 1. Results of ANOVAs performed on each life-history variable The analysis of pupal developmental rate used pupal weight as a covariate in order to remove any effects on developmental rate due to size differences Life-history trait Effect ANOVA P Pupal weight Temperature F 3,206 = Sex F 1,206 = Temperature sex F 3,206 = Larval developmental rate Temperature F 3,206 = Sex F 1,206 = Temperature sex F 3,206 = Pupal developmental rate Temperature F 3,205 = Sex F 1,205 = Temperature sex F 3,205 = Growth rate Temperature F 3,206 = Sex F 1,206 = Temperature sex F 3,206 = Fig. 2. The effect of temperature on life-history attributes of male (closed squares, dashed line) and female (open squares, solid line) H. bolina. The error bars represent 1 s.e. of each mean. Sample sizes for all plots are given in the growth rate plot, with female samples indicated above and male samples indicated below. The method of calculation of growth and developmental rates is given in the text.

7 72 D. J. Kemp Table 2. Initial and final sample sizes, final sex ratios, and percentage mortality for each rearing treatment used in the temperature and photoperiod experiments Initial sample sizes are given by the number of eggs in each treatment that hatched, and mortality was calculated as the proportion of this initial cohort that failed to pupate Experiment Treatment Sample sizes Sex ratio Mortality Initial Final (M : F) (%) 1. Temperature 21 C : C : C : C : Photoperiod 11.5-h light : h light : h light : Table 3. Relationship between size (forewing length [mm]; S) and weight (pupal weight [g]; W) for individuals reared in each temperature treatment, and for all treatments pooled Variables were log-transformed prior to conducting these analyses Temp. Regression s.e. R 2 F d.f. P ( C) slope 21 S = W , S = W , S = W , S = W , All S = W , temperature: Table 1). In the case of both developmental stages, males and females diverged most at the higher-temperature treatments, and converged at the lower-temperature treatments (Fig. 2). For example, there was no significant difference in either larval (F 1,36 = 1.3, P > 0.25) or pupal (F 1,36 = 0.653, P > 0.40) developmental rate between males and females in the 21 C treatment group, whereas overall sex effects in these attributes were highly significant. Pupal weight was significantly related to wing length in all four temperature treatments (Table 3), hence heavier pupae gave rise to adults with longer forewings. This relationship was linearised by log-transforming both variables, and the slopes of these regression lines were compared using ANCOVA. No significant difference was detected between slopes (F 3,202 = 0.98, P > 0.25), hence butterflies in all temperature treatments exhibited a similar winglength weight relationship. In addition, the slopes of regression lines calculated separately for males and females (temperature treatments pooled) were not significantly different (ANCOVA, F 1,206 = 0.40, P > 0.50). On this basis, observations from all treatments and both sexes were pooled, and the overall species winglength weight relationship calculated (Table 3). The slope of this regression line was significantly different ( = 0.001) from the isometric value of (99.9% confidence interval of regression slope = ). Wing length and pupal weight were therefore related allometrically in these reared individuals, with proportionally greater allocation to wing length made by relatively larger animals. Photoperiod effects on juvenile development As was the case with temperature, developmental attributes responded to variation in daylength (Fig. 3). This factor significantly affected growth rates (F 2,42 = 6.2, P < 0.005), developmental rates (F 2,42 = 11.0, P < ) and size at maturity (F 2,42 = 7.3, P < 0.002). On

8 Life-history plasticity in Hypolimnas bolina 73 Fig. 3. The effect of photoperiod on the life-history attributes of H. bolina (sexes pooled). The developmental rate shown here pertains to the larval stage only. The error bars represent 1 s.e. of each mean, and sample sizes for all plots are given in the growth rate plot. The method of calculation of growth and developmental rates is given in the text.

9 74 D. J. Kemp Fig. 4. The state of ovarian development of individuals reared in three photoperiod treatments (11.5, 12.5 and 13.5 light hours). The ovarian development classes are explained in the text, and sample sizes are given in parentheses. average, individuals grew more slowly and matured at a smaller size with increasing daylengths (Fig. 3), which is in accordance with predictions drawn from the adaptive explanation. However, developmental rate was not proportionally related to photoperiod. Of the three treatment groups, individuals in the intermediate daylength group (12.5 h light) had the lowest rate of development. On average, these larvae took longer (mean developmental time = 26.0 ± 0.4 days) to reach a smaller size than those individuals reared in the 11.5-h light treatment (mean developmental time = 24.7 ± 0.3 days). Mortality in this experiment was higher than that observed in the temperature experiment (9.1 15%: Table 2). Also, all individuals in this experiment grew considerably more slowly and pupated at a smaller size than those reared in the most closely corresponding treatment of the temperature experiment (the 27 C treatment: Fig. 1). Effect of photoperiod on ovarian development As predicted, post-eclosion ovarian development of female H. bolina varied according to developmental photoperiod (Fig. 4). Whereas all (n = 9) adults reared under constant 13.5-h light photoperiod contained gravid ovaries after 10 days, females reared under 11.5 hours daylight had either totally regressed (n = 3) or partially developed (n = 2) ovaries. Individuals reared under 12.5 hours daylight were intermediate between these two extremes. The average length of the basal (most mature) oocyte differed significantly between the three photoperiod treatments (F 2,19 = 16.9, P < 0.001). Ovarian development was not related to size (forewing length) within either the 11.5-h light (Spearman r s = 0.58, n = 5, P > 0.25) or 12.5-h light (Spearman r s = 0, n = 7, P = 1.0) treatments, which shows that larger individuals may not require more time for their ovaries to mature. All adults in the 11.5-h and 12.5-h light treatments were classified as having an extensive fat body, whilst all individuals in the 13.5-h treatment were classified as having moderate fat bodies. These findings are consistent with the hypothesis that photoperiod acts as a cue for diapause induction in this species. Discussion Although based on limited experiments, this investigation clearly demonstrated that (a) both temperature and photoperiod cause phenotypic plasticity in juvenile developmental traits in H. bolina, and (b) variation in rearing photoperiod influences the timing of oocyte development

10 Life-history plasticity in Hypolimnas bolina 75 in the adults. This suggests that plasticity in the size of adult H. bolina is not a wholly incidental effect of temperature variation (as predicted by the incidental effects hypothesis), and that at least some of the variation observed in field populations will relate to seasonal changes in daylength. Furthermore, these results suggest that photoperiod may be used by this tropical species as a cue for seasonal synchronisation, and for the timing of reproductive diapause induction. The limitations to these conclusions are that (a) the relative importance of temperature and photoperiod as determinants of life-history plasticity in the field cannot be readily partitioned, (b) any interaction between the effects of these environmental factors cannot be explored. Variation in ovarian development When reared under short-day conditions (11.5-h photoperiod), adult H. bolina clearly delayed their ovarian development (relative to that of long day individuals). This result appears similar to the response of many adult-diapausing temperate-zone butterfly species that use daylength as a cue for diapause induction (refer to references in Introduction). However, this preliminary result cannot be taken as evidence that photoperiod is the only, or even the primary, environmental cue for the induction of reproductive dormancy in this species. Rearing temperature often modifies the photoperiodic effect in many insect species with photoperiodically induced diapause (Danks 1987). Furthermore, both temperature and rainfall have been shown to affect seasonal polyphenism in tropical satyrid butterflies (Roskam and Brakefield 1998). In any case, it is clear that rearing photoperiod does exert some influence over the timing of adult reproduction, which is a notable result, since daylength may not necessarily provide the most appropriate cue for seasonal adversity in tropical areas (see Introduction). This results of this study lead to the proposal that diapause may be induced in H. bolina by rearing juveniles under photoperiods less than approximately 12.5 hours (although this may be modified by temperature: Danks 1987). This proposal is supported by the observed breeding phenology of H. bolina in the field (Fig. 1). In Cairns, daylength (including civil twilight) drops below the 12.5-h threshold in early mid March. If diapause is indeed induced by photoperiod as suggested by these rearing results, then we would expect adults to start eclosing in a state of reproductive diapause some time from early April onwards (or earlier depending on the sensitive stage of the larvae). This is indeed the time at which individuals begin entering overwintering sites in Cairns (Fig. 1), which strongly supports the conclusion that photoperiod drives the induction of diapause in this species. However, further work is probably required to confirm that short day adults, as reared in this study, actually enter a state of true reproductive diapause. Variation in juvenile developmental traits The relationship between temperature and development of juvenile H. bolina conforms to the generally recognised rule for ectothermic organisms (Atkinson 1994; Berrigan and Charnov 1994). In a recent review, Atkinson (1994) reported that lower temperature led to slower growth and later maturation at a larger size in approximately 80% of over 100 studied ectotherms. There is ample representation of the low temperature large size relationship among both tropical (e.g. Jones 1992) and temperate-zone (e.g. Pullin 1986) species of butterfly (although see James 1987 on migrating Australian nymphalid species). Traditionally, this relationship has been interpreted as a simple physiological cause-and-effect regime, whereby the life histories of ectothermic organisms are passively dictated by their thermal environment. However, recent research has indicated that several temperate-zone butterfly species have the ability to actively control their rate of growth (Nylin 1994 and references therein; Leimar 1996). These species increase their growth rate in response to shorter photoperiods that indicate progressively later dates in the breeding season, which allows them to develop more quickly whilst still maturing at a relatively large size (Nylin 1994 and references therein; Nylin et al. 1995, 1996; Leimar 1996; Gotthard et al. 1999). On this basis, Nylin (1994) suggested that life histories and developmental pathways may be more profitably viewed as chosen by the organism rather than dictated by the

11 76 D. J. Kemp environment. However, it is not clear whether tropical butterfly species such as H. bolina possess the ability to vary their growth rate in this manner. The finding that juveniles reared under different constant temperatures exhibited clear sexual dimorphism in developmental attributes corroborates field-based phenotypic observations, in which female H. bolina are on average significantly larger than males (Kemp, unpublished data). This is the general rule in invertebrates (Wiklund and Karlsson 1988; Nylin and Gotthard 1998). Interestingly, however, sexual dimorphism extended to the actual developmental responses to temperature variation (Fig. 2). This result suggests that wet-season butterflies should be proportionally more sexually dimorphic than their dry-season counterparts. However, at least in terms of size (forewing length), this regime is not supported by field observations, which indicate seasonally consistent levels of sexual size dimorphism (Kemp, unpublished data). Investigation of sexual differences in phenotypic response to photoperiod (which were not available here) may shed further light on this point. Although ectotherms are expected to exhibit developmental variation due to direct physiological effects of temperature on development and growth (Atkinson 1994; Berrigan and Charnov 1994), plasticity in response to changes in daylength is not necessarily expected. In insects, this type of plasticity has been generally recognised as a clear and unambiguous seasonal adaptation (Danilevskii 1965; Tauber et al. 1986; Danks 1987; Nylin and Gotthard 1998). On this basis, the presence of daylength-mediated variation in juvenile development supports the possibility that size plasticity in H. bolina is adaptive. Similar developmental and morphological variation shown by butterflies in response to photoperiod has been interpreted adaptively (Rienks 1985; James 1987; Jones 1992). However, although size variation in H. bolina occurred in the direction that would be predicted by an adaptive hypothesis (in the direction of larger overwintering adults), there are other explanations that could also make this prediction. For instance, there is some suggestion that, under field conditions, the larvae of this species feed only during the night (McCubbin 1971). If juveniles fed exclusively, or more often, during dark hours in this study, then this could account for the observed variation in life-history traits on the basis of varying rearing photoperiod. Although this discussion has focused on aspects of within-rearing developmental variation, it is important to note that developmental traits also varied between the rearing experiments. Individuals in the photoperiod experiment grew more slowly and pupated at a smaller size than did those reared under similar conditions in the temperature experiment. Mortality was also generally higher in the photoperiod experiment (Table 2). Since rearing methods were similar, the most likely explanation for this difference is that different larval hosts used in the experiments differed in terms of suitability for larval growth, perhaps due to differences in leaf water content or chemistry (Scriber and Slansky 1981). This is an interesting result, since S. nodiflora, the plant used in the photoperiod experiment, is apparently actively favoured over other foodplants (but not including A. gangetica) by ovipositing females in tropical north Queensland (Kemp 1998). However, among the Lepidoptera, plants that are apparently preferred for oviposition may not necessarily sustain the best rate of larval growth (Thompson and Pellmyr 1991). If the between-rearing developmental variation seen here does relate to differences in suitability of larval foodplants, then this suggests another likely source of phenotypic variation in the body size of individuals in the field. Although plasticity under this scenario obviously arises from a constraint, the ability to pupate at small size in response to resource limitation may itself be considered adaptive (Nylin 1994). Plasticity in body size of field populations of H. bolina may therefore represent the end product of a complex interaction between various adaptive and non-adaptive mechanisms; however, the extent to which this plasticity is adaptive may be best defined experimentally (see Gotthard and Nylin 1995). In summary, this investigation has shown that aspects of the development and life history of this tropical species, including the timing of adult reproduction, are indeed influenced by rearing photoperiod, which suggests that H. bolina may provide an excellent model species for further investigations into the evolution of optimal life histories in the tropics.

12 Life-history plasticity in Hypolimnas bolina 77 Acknowledgments Thanks to Dr Jamie Seymour (James Cook University) for suggestions on an earlier version of this manuscript and for allowing the use of constant-environment cabinets. An anonymous reviewer also provided a particularly helpful critique. This study was supported by an Australian Postgraduate Research Award. References Andersen, N. M. (1993). The evolution of wing polymorphism in water striders (Gerridae): a phylogenetic approach. Oikos 67, Atkinson, D. (1994). Temperature and organism size a biological law for ectotherms? Advances in Ecological Research 25, Berrigan, D., and Charnov, E. L. (1994). Reaction norms for age and size at maturity in response to temperature: a puzzle for life historians. Oikos 70, Braby, M. F. (1994). Phenotypic variation in adult Mycalesis Hubner (Lepidoptera : Nymphalidae : Satyrinae) from the Australian wet dry tropics. Journal of the Australian Entomological Society 33, Braby, M. F. (1995). Reproductive seasonality in tropical satyrine butterflies: strategies for the dry season. Ecological Entomology 20, Chaplin, S. B., and Wells, P. H. (1982). Energy reserves and metabolic expenditures of monarch butterflies overwintering in southern California. Ecological Entomology 7, Clarke, C., and Sheppard, P. M. (1975). The genetics of the mimetic butterfly Hypolimnas bolina. Philosophical Transactions of the Royal Society of London (B) 272, Clarke, S. H., and Platt, A. P. (1969). Influence of photoperiod on development and larval diapause in the viceroy butterfly, Limenitis archippus. Journal of Insect Physiology 15, Danilevskii, A. S. (1965). Photoperiodism and Seasonal Development of Insects. (Oliver & Boyd: Edinburgh.) Danks, H. V. (1987). Insect Dormancy: an Ecological Perspective. Biological Survey of Canada Monograph Series No. 1. (Biological Survey of Canada: Ottawa.) Denlinger, D. L. (1986). Dormancy in tropical insects. Annual Review of Entomology 31, Gordon, I. J., and Smith, D. A. S. (1998). Body size and colour-pattern genetics in the polymorphic mimetic butterfly Hypolimnas misippus (L.). Heredity 80, Gotthard, K., and Nylin, S. (1995). Adaptive plasticity and plasticity as an adaptation: a selective review of plasticity in animal morphology and life history. Oikos 74, Gotthard, K., Nylin, S., and Wiklund, C. (1999). Seasonal plasticity in two satyrine butterflies: statedependent decision making in relation to daylength. Oikos 84, Harada, T., and Numata, H. (1993). Two critical day lengths for the determination of wing forms and the induction of adult diapause in the water strider, Aquarius paludum. Naturwissenschaften 80, Hazel, W. N., and West, D. A. (1983). The effect of larval photoperiod on pupal colour and diapause in swallowtail butterflies. Ecological Entomology 8, Holloway, G. J. (1993). Phenotypic variation in colour pattern and seasonal plasticity in Eristalis hoverflies (Diptera : Syrphidae). Ecological Entomology 18, James, D. G. (1987). Effects of temperature and photoperiod on the development of Vanessa kershawi McCoy and Junonia villida Godart (Lepidoptera : Nymphalidae). Journal of the Australian Entomological Society 26, Jones, R. E. (1987). Reproductive strategies for the seasonal tropics. Insect Science and its Application 8, Jones, R. E. (1992). Phenotypic variation in Australian Eurema species. Australian Journal of Zoology 40, Jones, R. E., and Rienks, J. (1987). Reproductive seasonality in the tropical genus Eurema (Lepidoptera : Pieridae). Biotropica 19, Kemp, D. J. (1998). Oviposition behaviour of post-diapause Hypolimnas bolina (L.) (Lepidoptera : Nymphalidae) in tropical Australia. Australian Journal of Zoology 46, Leimar, O. (1996). Life history plasticity: influence of photoperiod on growth and development in the common blue butterfly. Oikos 76, McCubbin, C. (1971). Australian Butterflies. (Nelson: Melbourne.) Nylin, S. (1989). Effects of changing photoperiods in the life cycle regulation of the comma butterfly, Polygonia c-album (Nymphalidae). Ecological Entomology 14,

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