Evolution and biogeography of the endemic Roucela complex (Campanulaceae: Campanula) in the Eastern Mediterranean

At the intersection of geological activity, climatic fluctuations, and human pressure, the Mediterranean Basin – a hotspot of biodiversity – provides an ideal setting for studying endemism, evolution, and biogeography. Here, we focus on the Roucela complex (Campanula subgenus Roucela), a group of 13 bellflower species found primarily in the eastern Mediterranean Basin. Plastid and low‐copy nuclear markers were employed to reconstruct evolutionary relationships and estimate divergence times within the Roucela complex using both concatenation and species tree analyses. Niche modeling, ancestral range estimation, and diversification analyses were conducted to provide further insights into patterns of endemism and diversification through time. Diversification of the Roucela clade appears to have been primarily the result of vicariance driven by the breakup of an ancient landmass. We found geologic events such as the formation of the mid‐Aegean trench and the Messinian Salinity Crisis to be historically important in the evolutionary history of this group. Contrary to numerous past studies, the onset of the Mediterranean climate has not promoted diversification in the Roucela complex and, in fact, may be negatively affecting these species. This study highlights the diversity and complexity of historical processes driving plant evolution in the Mediterranean Basin.     

The inheritance of modifiers conferring self-fertility in the partially self-incompatible perennial, Campanula rapunculoides L. (Campanulaceae)

The role of partial self-incompatibility in plant breeding system evolution has received little attention. Here, we examine the genetic basis of modifiers conferring self-fertility in the creeping bellflower, Campanula rapunculoides L. (Campanulaceae), a partially self-incompatible herb. A survey of 35 individuals from two natural populations indicates that 45% of them are strongly self-incompatible, 40% intermediately self-incompatible, and 15% weakly self-incompatible and that some plants show a strong breakdown in self-incompatibility over floral age. We generated 101 F1 families by random crossing among 31 parental plants and estimated the heritability of self-fertility in day 1 and day 4 female-phase flowers, the genetic correlation between day 1 and day 4 self-fertility, and the coefficient of additive genetic variance of self-fertility. We use linear regression and data from additional crosses to examine whether there are significant maternal effects in the expression of self-fertility. We use Fain's test to determine if a major gene influences self-fertility and, finding no evidence, use data from additional crosses on an F2 generation to estimate the mean number and dominance of genes conferring self-fertility. These analyses indicate that the heritability (h2) of self-fertility is 0.24 in day 1 female-phase flowers and 0.44 in day 4 flowers, self-fertility is primarily additive but shows some recessive effects, and self-fertility is estimated to be controlled by four genetic factors. In addition, we have evidence that there may be maternal effects for self-fertility, especially for weakly self-incompatible plants. The significance of these results in the context of mating system evolution is discussed.     

Genotype–environment interaction and the ontogeny of diet-induced phenotypic plasticity in size and shape of Melanoplus femurrubrum (Orthoptera: Acrididae)

The developmental origin of phenotypic plasticity in morphological shape can be attributed to environment-specific changes in growth of overall body size, localized growth of a morphological structure or a combination of both. I monitored morphological development in the first four nymphal instars of grasshoppers (Melanoplus femurrubrum) raised on two different plant diets to determine the ontogenetic origins of diet-induced phenotypic plasticity and to quantify genetic variation for phenotypic plasticity. I measured diet-induced phenotypic plasticity in body size (tibia length), head size (articular width and mandible depth) and head shape (residual articular width and residual mandible depth) for grasshoppers from 37 full-sib families raised on either a hard plant diet (Lolium perenne) or a soft plant diet (Trifolium repens). By the second to third nymphal instar, grasshoppers raised on a hard plant diet had significantly smaller mean tibia length and greater mean residual articular width (distance between mandibles adjusted for body size) compared with full-sibs raised on a soft plant diet. However, there was no significant phenotypic plasticity in mean unadjusted articular width and mandible depth, and in mean residual mandible depth. At the population level, development of diet-induced phenotypic plasticity in grasshopper head shape is mediated by plastic changes in allocation to tissue growth that maintain growth of head size on hard, low-nutrient diets while reducing growth of body size. Within the population, there was substantial variation in the plasticity of growth trajectories since different full-sib families developed phenotypic plasticity of residual articular width through different combinations of head and body size growth. Genetic variation for diet-induced phenotypic plasticity of residual articular width, residual mandible depth and tibia length, as estimated by genotype–environment interaction, exhibited significant fluctuation through ontogeny (repeated measures MANOVA , family × plant × instar, P < 0.01). For example, there was significant genetic variation for phenotypic plasticity of residual articular width in the third nymphal instar, but not earlier or later in ontogeny. The observed patterns of genetic variation are discussed with reference to short-term constraints and the evolution of phenotypic plasticity.     

Reproduction inCampanula afra (Campanulaceae): Mating system and the role of the pollen collecting hairs

The aim of the present investigation is to compare two geographically well separated populations ofC. afra with different mating systems with emphasis on (1) factors controlling the length of the male phase with special attention paid on the pollen collecting hairs, (2) crossing ability among populations with different mating systems; shouldC. afra be regarded as one or several species?, and (3) correlations between pollen germinability and mating system; how does pollen germinability vary with anthesis and stigma development? From the results it can be concluded that the length of the male phase and the development of the stigma depend on the stimulation of the pollen collecting hairs rather than on the amount of pollen removed. Furthermore, the length of the male phase was found to be correlated with the mating system, i.e., in the population with a mixed mating system, the differences between treated and untreated flowers were less pronounced than in the population being obligately outcrossing. I also found correlations between mating system and pollen germinability, where the population with a mixed mating system had a high germinability at stigma receptivity, whereas for the xenogamous population, the germinability approached zero at stigma development. No crossing incompatibility was found between the two populations ofCampanula afra in any of the combinations.     

Short-term effects of fire and competition on growth and plasticity of the yellow pitcher plant,Sarracenia alata (Sarraceniaceae)

Although recurrent fires are widely assumed to reduce competitive interference of plants of pine savannas, rarely has this assumption been tested explicitly. This 2-yr study reports on the interactive effects of fire and neighbors on short-term growth responses and plasticity in allocation patterns of a carnivorous plant, the yellow pitcher plant, Sarracenia alata. This species relies upon pitfall traps (pitchers) to attract and capture insects to obtain nutrients. Neighbors reduced the growth rate of individual ramet transplants (phytometers) in one but not both years of the study. The effect of neighbors on total (i.e., both above- and belowground) productivity of phytometers was not reduced by a winter fire. Neighbors had a greater effect on large plants than on small plants. Although fire did not affect the growth rate of phytometers in the short term, allocation patterns were greatly altered by both neighbors and fire. Allocation to pitchers increased at the expense of belowground organs following fire and in the absence of neighbors at the unburned site. Results of the current study suggest that adult pitcher plants may tolerate competition from neighboring vegetation by reducing allocation to costly pitchers during years without fire.     

Pollen dispersal and gene flow within and into a population of the alpine monocarpic plant Campanula thyrsoides

Background and Aims

Gene flow by seed and pollen largely shapes the genetic structure within and among plant populations. Seed dispersal is often strongly spatially restricted, making gene flow primarily dependent on pollen dispersal within and into populations. To understand distance-dependent pollination success, pollen dispersal and gene flow were studied within and into a population of the alpine monocarpic perennial Campanula thyrsoides.

Methods

A paternity analysis was performed on sampled seed families using microsatellites, genotyping 22 flowering adults and 331 germinated offspring to estimate gene flow, and pollen analogues were used to estimate pollen dispersal. The focal population was situated among 23 genetically differentiated populations on a subalpine mountain plateau (<10 km²) in central Switzerland.

Key Results

Paternity analysis assigned 110 offspring (33·2 %) to a specific pollen donor (i.e. ‘father’) in the focal population. Mean pollination distance was 17·4 m for these offspring, and the pollen dispersal curve based on positive LOD scores of all 331 offspring was strongly decreasing with distance. The paternal contribution from 20–35 offspring (6·0–10·5 %) originated outside the population, probably from nearby populations on the plateau. Multiple potential fathers were assigned to each of 186 offspring (56·2 %). The pollination distance to ‘mother’ plants was negatively affected by the mothers'' degree of spatial isolation in the population. Variability in male mating success was not related to the degree of isolation of father plants.

Conclusions

Pollen dispersal patterns within the C. thyrsoides population are affected by spatial positioning of flowering individuals and pollen dispersal may therefore contribute to the course of evolution of populations of this species. Pollen dispersal into the population was high but apparently not strong enough to prevent the previously described substantial among-population differentiation on the plateau, which may be due to the monocarpic perenniality of this species.     

Pollination of Campanula rapunculus L. (Campanulaceae): How much pollen flows into pollination and into reproduction of oligolectic pollinators?

We studied an isolated population of Campanula rapunculus and two oligolectic bee species of Chelostoma (Megachilidae), their main pollinators. The population of C. rapunculus consisted of 2808 plants. Measurements of pollen flow showed that 3.7% of the pollen produced by a flower contribute to pollination, 95.5% was collected by bees for their offspring and 0.8% remained on the styles. Pollen analyses of brood cells of Chelostoma rapunculi revealed that females collected on average 4.9 million Campanula pollen to rear one bee. We calculated that approximately 1588 bees of this species could have been reared at the study site during the studied season. The amount of potentially viable pollen deposited on stigmas was 3.6 to 10.7 times higher than the number of ovules. We discuss morphological features of the flowers which may lower the pollen removal rate per bee visit and consequently cause a high visitation and pollination rate.The study was supported by a joint project CAPES / DAAD (Probral 112/00). We thank NaBu (Naturschutzbund) Bonn and the Untere Landschaftsbehörde Siegburg for the permission to work at the Natural Reserve Dünstekoven.     

Parental effects in a partially self-incompatible herb Campanula rapunculoides L. (Campanulaceae): influence of variation in the strength of self-incompatibility on seed set and progeny performance

We employ a full reciprocal diallel design between 10 parental plants that differed in their strength of self-incompatibility (SI; strong, intermediate, and weak) to examine parental effects on seed set and 10 components of fitness of progeny performance in Campanula rapunculoides. We perform ANOVAs to separate the influence of the strength of SI and the identity of the maternal and paternal parent on family performance. We calculate the phenotypic and genetic correlations between traits to determine potentially evolutionary constraints. Finally, we employ maximum likelihood methods to estimate the components of quantitative genetic variance, as defined by Cockerham and Weir in their BioModel c. Our most significant finding is that weak SI plants have high outcrossed seed set as maternal parents. We argue that direct or pleiotropic effects of modifiers of SI probably cause this. Second, we find that extranuclear interactions, as defined by the BioModel, have strong effects on seed set and several vegetative and flowering traits. These findings indicate that some maternal plants selectively provision seeds sired by specific paternal donors and that some of this variation appears to be associated with modifiers of the strength of SI. We find other sources of significant quantitative genetic variation for all of the traits we examine and discuss the possible role these play in the evolution of the reproductive system. Taken together, our findings show that variation in the strength of SI may influence levels of quantitative genetic variation that, in turn, can influence the reproductive success of individuals in C. rapunculoides     

Secondary pollen presentation and the temporal dynamics of stylar hair retraction and style elongation in Campanula trachelium (Campanulaceae)

To increase the accuracy of pollen capture and transfer by pollinators some plant species have developed secondary pollen presentation structures. Because the presence of secondary pollen presentation structures at the pistil may reduce the spatial separation between the sexual functions and increase the risk of self‐interference and selfing, temporal segregation of the sexual organs, triggered by visiting insects, can be expected to occur. We investigated secondary pollen presentation and the temporal dynamics of the sexual phases in combination with the physiological self‐incompatibility system in Campanula trachelium, a protandrous insect‐pollinated herb. Stylar hair retraction (male function) and curling of the stigmatic lobes (female function) were modelled using Gompertz growth functions. Finally, we performed pollination experiments in the lab and field to assess seed set and pollen limitation under natural conditions. About 68% of the total pollen load was captured by stylar hairs. Manual manipulation of the stylar hairs, mimicking pollinator visitation, significantly shortened the male phase and accelerated the female phase, resulting in a significant decline in temporal overlap between the two sexual functions. Conversely, when pollinators and/or manual manipulations were lacking, the male phase was substantially prolonged and sexual overlap was maximal. This suggests that spreading of the sexual phases and thus the risk of sexual interference are largely determined by the interaction between stylar hairs and visiting pollinators. Natural seed set was high and not pollinator limited. Overall, these results indicate that secondary pollen presentation and partial protandry resulted in efficient pollen capture, transfer and deposition.     

Rapid adaptation to a novel light environment: The importance of ontogeny and phenotypic plasticity in shaping the visual system of Nicaraguan Midas cichlid fish (Amphilophus citrinellus spp.)

Colonization of novel habitats is typically challenging to organisms. In the initial stage after colonization, approximation to fitness optima in the new environment can occur by selection acting on standing genetic variation, modification of developmental patterns or phenotypic plasticity. Midas cichlids have recently colonized crater Lake Apoyo from great Lake Nicaragua. The photic environment of crater Lake Apoyo is shifted towards shorter wavelengths compared to great Lake Nicaragua and Midas cichlids from both lakes differ in visual sensitivity. We investigated the contribution of ontogeny and phenotypic plasticity in shaping the visual system of Midas cichlids after colonizing this novel photic environment. To this end, we measured cone opsin expression both during development and after experimental exposure to different light treatments. Midas cichlids from both lakes undergo ontogenetic changes in cone opsin expression, but visual sensitivity is consistently shifted towards shorter wavelengths in crater lake fish, which leads to a paedomorphic retention of their visual phenotype. This shift might be mediated by lower levels of thyroid hormone in crater lake Midas cichlids (measured indirectly as dio2 and dio3 gene expression). Exposing fish to different light treatments revealed that cone opsin expression is phenotypically plastic in both species during early development, with short and long wavelength light slowing or accelerating ontogenetic changes, respectively. Notably, this plastic response was maintained into adulthood only in the derived crater lake Midas cichlids. We conclude that the rapid evolution of Midas cichlids’ visual system after colonizing crater Lake Apoyo was mediated by a shift in visual sensitivity during ontogeny and was further aided by phenotypic plasticity during development.     

Males are cheaper,or the extreme consequence of size/age‐dependent sex allocation: sexist gender diphasy in Fritillaria montana (Liliaceae)

The bulbous geophyte Fritillaria montana is partially self‐compatible and capable of switching gender. Small flowering plants produce only single male flowers, but larger plants produce hermaphrodite or, rarely, male and hermaphrodite flowers. Eight populations in peninsular Italy were sampled to determine the frequency and size distributions of male and hermaphrodite plants, and to determine the relationship of plant size to male and hermaphrodite flower production. Male plants were significantly smaller than hermaphrodites and made up 14.5–47.8% (100% in one small population) of flowering plants within populations. There were no significant differences in male fitness among female‐sterile and hermaphrodite flowers, as they both possessed full and comparable fertilizing power. Therefore, the gender variation observed in F. montana is likely to depend on resource‐dependent sex allocation. From an evolutionary perspective, we highlight the occurrence of similar mechanisms of gender variation in other representatives of the order Liliales. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 323–333.     

Development of distylous flowers and investment of biomass in male and female function in Palicourea padifolia (Rubiaceae)

Knowledge of developmental pathways for achieving differences in style and anther heights, in concert with those of ancillary features accompanied with data in regard to biomass investment to male and female function, provide an excellent opportunity for examining the developmental correlations between primary and ancillary floral traits so as to understand the evolution of heterostyly. The ontogenetic relationships between bud length and anther height and between bud length and style height, and between bud length versus bud width, anther length, and number of pollen grains per anther for long-styled (LS) and short-styled (SS) morphs of P. PADIFOLIA are described. We also described the ontogenetic biomass allocation to male and female function and to corolla with elongation of buds harvested at regular intervals. We observed an early termination of stylar growth in SS buds, whereas LS styles steadily increased in size. Morph differences for relative growth rates were significant for anther height, anther length, and pollen number but not for bud width. Bud width and anther length had a negative allometric relationship with bud elongation. The relationship between bud length and number of pollen grains per anther was positive and morph differences in pollen number were detected at later stages of development. An increase in corolla mass involved a disproportionate allocation to the female function in SS flowers and male allocation was similar for the two morphs over the course of development. Our results are consistent with theoretical and empirical data for distylous species with an approach herkogamous ancestor, and with the more general hypothesis of ontogenetic lability of heterostyly, in which morph differences in style and anther heights are achieved in various ways. Variations observed in sexual investment between floral morphs suggest differences in sex expression during flower development.     

Hidden floral adaptation to nocturnal moths in an apparently bee‐pollinated flower,Adenophora triphylla var. japonica (Campanulaceae)

• The discrepancy between observed flower visitors and those predicted based on floral phenotype has often cast doubt on the pollination syndrome concept. Here we show that this paradox may be alleviated by gaining better knowledge of the contributions of different flower visitors to pollination and the effects of floral traits that cannot be readily perceived by humans in Adenophora triphylla var. japonica. The blue, bell‐shaped and pendant flowers of A. triphylla appear to fit a bee pollination syndrome. In contrast to this expectation, recent studies show that these flowers are frequented by nocturnal moths.
• We compared the flower visitor fauna, their visitation frequency and their relative contributions to seed set between day and night in two field populations of A. triphylla in Japan. We also determined the floral traits associated with temporal changes in the visitor assemblage, i.e. the timing of anthesis, the timing of changes in the sexual phase and the diel pattern of nectar production.
• While A. triphylla flowers were visited by both diurnal and nocturnal insects, the results from pollination experiments demonstrate that their primary pollinators are nocturnal settling‐moths. Moreover, the flowers opened just after sunset, changed from staminate to pistillate phase in successive evenings and produced nectar only during the night, which all conform to the activity of nocturnal/crepuscular moths.
• Our study illustrates that the tradition of stereotyping the pollinators of a flower based on its appearance can be misleading and that it should be improved with empirical evidence of pollination performance and sufficient trait matching.

     

Testing the effects of heterozygosity on growth rate plasticity in the seaweed Gracilaria chilensis (Rhodophyta)

Heterozygosity has been positively associated with fitness and population survival. However, the relationship between heterozygosity and adaptive phenotypic plasticity (i.e., plasticity which results in fitness homeostasis or improvement in changing environments) is unclear and has been poorly explored in seaweeds. In this study, we explored this relationship in the clonal red seaweed, Gracilaria chilensis by conducting three growth rate plasticity experiments under contrasting salinity conditions and by measuring heterozygosity with five microsatellite DNA markers. Firstly, we compared growth rate plasticity between the haploid and diploid phases. Secondly, we compared growth rate plasticity between diploids with different numbers of heterozygous loci. Finally, we compared growth rate plasticity between diploid plants from two populations that are expected to exhibit significant differences in heterozygosity. We found that, (i) diploids displayed a higher growth rate and lower growth rate plasticity than haploids, (ii) diploids with a higher number of heterozygous loci displayed lower growth rate plasticity than those exhibiting less heterozygosity, and (iii) diploid sporophytes from the population with higher heterozygosity displayed lower growth rate plasticity than those with lower heterozygosity. Accordingly, this study suggests that heterozygosity is inversely related to growth rate plasticity in G. chilensis. However, better genetic tools in seaweeds are required for a more definitive conclusion on the relationship between heterozygosity and phenotypic plasticity.     

Multigenerational effects of inbreeding in Cucurbita pepo ssp. texana (Cucurbitaceae)

The shape of the fitness function relating the decline in fitness with coefficient of inbreeding (f) can provide evidence concerning the genetic basis of inbreeding depression, but few studies have examined inbreeding depression across a range of f using noncultivated species. Futhermore, studies have rarely examined the effects of inbreeding depression in the maternal parent on offspring fitness. To estimate the shape of the fitness function, we examined the relationship between f and fitness across a range off from 0.000 to 0.875 for components of both male and female fitness in Cucurbita pepo ssp. texana. Each measure of female fitness declined with f, including pistillate flower number, fruit number, seed number per fruit, seed mass per fruit, and percentage seed germination. Several aspects of male fitness also declined with f, including staminate flower number, pollen number per flower, and the number of days of flowering, although cumulative inbreeding depression was less severe for male (0.34) than for female function (0.39). Fitness tended to decline linearly with f between f = 0.00 and f = 0.75 for most traits and across cumulative lifetime fitness (mean = 0.66), suggesting that individual genes causing inbreeding depression are additive and the result of many alleles of small effect. However, most traits also showed a small reduction in inbreeding depression between f = 0.75 and f = 0.875, and evidence of purging or diminishing epistasis was found for in vitro pollen-tube growth rate. To examine inbreeding depression as a maternal effect, we performed outcross pollinations on f = 0.0 and f = 0.5 mothers and found that depression due to maternal inbreeding was 0.07, compared to 0.10 for offspring produced through one generation of selfing. In at least some families, maternal inbreeding reduced fruit number, seed number and mass, staminate flower number, pollen diameter, and pollen-tube growth rate. Collectively these results suggest that, while the fitness function appears to be largely linear for most traits, maternal effects may compound the effects of inbreeding depression in multigenerational studies, though this may be partially offset by purging or diminishing epistasis.     

Phenotypic variation in colour pattern and seasonal plasticity in Eristalis hoverflies (Diptera: Syrphidae)

Abstract.

• 1 An examination of phenotypic variation in colour pattern was carried out on four Eristalis hoverfly species using museum material.
• 2 The amount of phenotypic variation varied substantially among the species with E.arbustorum being the most variable. The other species showed a wide colour pattern range but less variation within that range (E.abusivus and E.nemorum), or a narrow range of colour variation (E.horticola).
• 3 Sexual colour dimorphism was apparent in all four species, but most pronounced in E.abusivus and E.nemorum.
• 4 There were good phenotype-season relationships shown by both sexes in all species, except for female E.abusivus and E.nemorum, with paler insects being more abundant during the warmer summer months.
• 5 Female, but not male, E.arbustorum collected at inland sites were on average paler than those collected at coastal sites. This observation is considered with respect to temperature during the developmental stages.
• 6 The function of colour plasticity in hoverflies is discussed with reference to the need to maintain optimal thermal conditions for activity.

     

Effects of low-efficiency pollinators on plant fitness and floral trait evolution in <Emphasis Type="Italic">Campanula americana</Emphasis> (Campanulaceae)

Floral visitors vary in their pollination efficiency and their preferences for floral traits. If low-efficiency pollinators decrease the amount of pollen available to higher efficiency visitors, then low-efficiency visitors may actually have negative fitness consequences for the plants that they visit. We used experimental arrays in two populations to determine the floral preferences and the fitness effects of low-efficiency (or ugly) pollinators on Campanula americana. These ugly pollinators (halictid bees) preferentially visited flowers with pollen over flowers that had had their pollen removed. C. americana pollen color varies quantitatively from light tan to dark purple, and we found that natural variation in pollen color influenced the magnitude of halictid preferences for flowers with pollen. In general, preferences for flowers with pollen were stronger when the ugly pollinators foraged in arrays of flowers with tan-colored pollen than in arrays with purple-colored pollen. When plants received few visits by efficient Bombus pollinators, visits by ugly pollinators significantly decreased siring success relative to plants where visits by ugly pollinators were prevented. In contrast, ugly pollinators did not influence siring success when higher efficiency pollinators were more abundant. Thus, the relationship between low-efficiency pollinators and the plants that they visit varies from commensalistic to antagonistic depending on the presence of other pollinators in the community. Our findings suggest that the negative fitness effects and floral preferences of low-efficiency or ugly pollinators may contribute to the maintenance of a pollen color polymorphism in C. americana.     

Seasonal plasticity in growth and development of the speckled wood butterfly, Pararge aegeria (Satyrinae)

Regulation of growth and development by photoperiod was studied in a population of the speckled wood butterfly, Purarge aegeria L. (Lepidoptera: Satyrinae), from southern Sweden. Individuals were reared in a range of photoperiodic regimes (9L. to 22L) and temperatures (13°C to 21° C). Plasticity was found for important life-history traits- generation time, growth rate and final weight and seasonal regulation of development in response to photoperiod was found to occur at two levels. Purarge aegeria hibernates as a third instar larva or in the pupal stage, cantering one of four major developmental pathways in response to photoperiod: (1) direct development in both the larval and pupal stages, (2) pupal winter diapause with or (3) without a preceding larval summer diapause, or (4) larval winter diapause. In addition to this high-level regulation of individual development, larval growth rate and pupal development rate also appear to be finally regulated by photoperiod within each major pathway. As photoperiods decreased from 22 h to 17 h at 17° C, growth rate among directly developing larvae increased progressively, as was the case for larva? developing according to a univoltine life cycle from 17 h to 14 h. At two photoperiods, 13 h and 16 h (corresponding to shifts between major pathways), both larval and pupal development were extremely variable with the fastest individuals developing directly and the slowest developing with a diapause. This indicates a gradual nature of diapause itself, suggesting that the two level may not he fundamentally different.