The period gene and allochronic reproductive isolation in Bactrocera cucurbitae

Clock genes that pleiotropically control circadian rhythm and the time of mating may cause allochronic reproductive isolation in the melon fly Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Flies with a shorter circadian period (ca. 22 h of locomotor activity rhythm) mated 5 h earlier in the day than those with a longer circadian period (ca. 30 h). Mate-choice tests demonstrated significant pre-mating isolation between populations with short and long circadian periods. Pre-mating isolation did not occur when the mating time was synchronized between the two populations by photoperiodic controls, indicating that reproductive isolation is due to variations in the time of mating and not any unidentified ethological difference between the two populations. We cloned the period (per) gene of B. cucurbitae that is homologous to the per gene in Drosophila. The relative level of per mRNA in the melon fly exhibited a robust daily fluctuation under light : dark conditions. The fluctuation of per expression under dark : dark conditions is closely correlated to the locomotor rhythm in B. cucurbitae. These results suggest that clock genes can cause reproductive isolation via the pleiotropic effect as a change of mating time.     

GENETIC CORRELATIONS BETWEEN LIFE-HISTORY AND BEHAVIORAL TRAITS CAN CAUSE REPRODUCTIVE ISOLATION

Reproductive isolation may often evolve as an indirect (pleiotropic) consequence of populations adapting to different environments or habitats. For example, niches that are temporally or seasonally offset can select for organisms with different developmental characteristics. These developmental differences can inadvertently cause reproductive isolation by a variety of means including shifts in mating activity patterns. Here, we show a genetic correlation between a life-history trait (developmental period) and a behavioral trait (time of mating) that causes significant premating isolation in the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae). Fly lines selected for short and long developmental periods differ in their preferred times of mating during the evening. This difference translates into significant prezygotic isolation, as measured by mate choice tests. If the time of mating between two populations differed more than one hour, the isolation index was significantly higher than zero. These indicate that premating isolation can be established if the developmental period is divergently selected for. If such genetic correlations are ubiquitous in many organisms, multifarious divergent selection for life-history traits would often accelerate the evolution of reproductive isolation. We speculate that reproductive isolation may have been evolved via genetic correlations among time-related traits, for example, developmental period and time of mating, as in other organisms.     

A Tale of Two Morphs: Modeling Pollen Transfer,Magic Traits,and Reproductive Isolation in Parapatry

The evolution of the flower is commonly thought to have spurred angiosperm diversification. Similarly, particular floral traits might have promoted diversification within specific angiosperm clades. We hypothesize that traits promoting the precise positional transfer of pollen between flowers might promote diversification. In particular, precise pollen transfer might produce partial reproductive isolation that facilitates adaptive divergence between parapatric populations differing in their reproductive-organ positions. We investigate this hypothesis with an individual-based model of pollen transfer dynamics associated with heterostyly, a floral syndrome that depends on precise pollen transfer. Our model shows that precise pollen transfer can cause sexual selection leading to divergence in reproductive-organ positions between populations served by different pollinators, pleiotropically causing an increase in reproductive isolation through a “magic trait” mechanism. Furthermore, this increased reproductive isolation facilitates adaptive divergence between the populations in an unlinked, ecologically selected trait. In a different pollination scenario, however, precise pollen transfer causes a decrease in adaptive divergence by promoting asymmetric gene flow. Our results highlight the idea that magic traits are not “magic” in isolation; in particular, the effect size of magic traits in speciation depends on the external environment, and also on other traits that modify the strength of the magic trait''s influence on non-random mating. Overall, we show that the evolutionary consequences of pollen transfer dynamics can depend strongly on the available pollinator fauna and on the morphological fit between flowers and pollinators. Furthermore, our results illustrate the potential importance of even weak reproductive isolating barriers in facilitating adaptive divergence.     

DISRUPTIVE SELECTION ON HABITAT PREFERENCE AND THE EVOLUTION OF REPRODUCTIVE ISOLATION: AN EXPLORATORY EXPERIMENT

A. pair of replicate experiments was carried out to examine the role of habitat specialization in the process of sympatric speciation. The rationale of the experimental design was that disruptive selection for habitat preference can facilitate the process of sympatric speciation by reducing an antagonistic interaction between the processes of recombination and selection. Replicate populations of Drosophila melanogaster were subjected to disruptive selection for habitat preference and simultaneous selection for positive assortative mating, for a period of 46 generations. The experimental protocol simulated the introduction of a frugivorous fly population into a new environment that contained a mosaic of eight equally abundant habitats. Only two habitats were suitable to the flies, and these required opposite patterns of geotaxis, phototaxis, chemotaxis, and developmental time to be successfully located. At the beginning of each generation, flies were placed as pupae into the middle of a maze containing eight discrete habitats. To produce disruptive selection on habitat preference, only those flies that chose one or the other of two selected habitats were permitted to contribute gametes to the next generation. Flies from each of the selected habitats were cultured separately to simulate independent carrying capacities within each habitat. Because flies rarely mated until they assorted themselves into the available habitats, genes affecting habitat preference pleiotropically produced assortative mating between flies with similar habitat preference. A genetic marker was used to continuously monitor the level of potential gene flow between the flies derived from the two selected habitats. During the course of the experiment, there was a gradual increase in the philopatry of the flies, indicating the development of habitat specialization. The evolution of habitat specialization resulted in substantial reproductive isolation between the subpopulations utilizing each habitat resource.     

Comparison of two polymorphic sites in the clock gene cryptochrome in the Taiwan strain of the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae): a possible quick method to estimate the mating time of trapped invading flies

For successful sterile insect technique (SIT), synchronized copulation between invaded females and sterilized males is required. Understanding the mating time of the invaded strain is an aid in synchronizing and thus improving the effectiveness of SIT. We previously demonstrated a relationship between variation at two sites of a circadian clock gene cryptochrome (cry) (cry1212 and cry1865) and circadian behavior in the melon fly Bactrocera cucurbitae (Coquillett). Here we investigated the relationship in two other populations, Taiwan1 (T1) and Taiwan2 (T2), which may re-invade Okinawa. The results showed that T1 exhibited a lower frequency of the S-type allele, which was observed in early mating flies in the strains in Okinawa, than T2 at the site of cry1212. In addition, T1 showed a longer circadian period than T2. We also noted that the cry1212 site showed higher amino acid sequence conservation than cry1865 by comparing CRY1 among five insect species. These results suggest that genotyping of only the cry1212 site of trapped flies enables an immediate estimate of the mating time of the B. cucurbitae population from Taiwan and that cry1212 would be more likely to be involved in determining the mating time than cry1865.     

Genetic changes of life history and behavioral traits during mass-rearing in the melon Fly,Bactrocera cucurbitae (Diptera: Tephritidae)

Quantitative genetic studies for life history and behavioral traits are important in quality control for insect mass-rearing programs. Firstly, a brief history of quality control in mass-reared insects is described. Next, the differentiation of many traits of wild and mass-reared melon flies,Bactrocera cucurbitae, in Okinawa is reviewed, and the factors which have caused variation in these traits are considered. As artificial selection pressures are thought to be more important than inbreeding depression and genetic drift in the mass-reared strain of the Okinawan melon fly, two artificial selection experiments were conducted to evaluate genetic variations and genetic correlations among life history and behavioral traits. These are divergent selections for age at reproduction and for developmental period. The genetic relationship among 5 traits, i.e. longevity, age at reproduction, developmental period, circadian period, and time of mating was clarified and discussed in relation to genetic changes of traits during the mass-rearing. The results suggest that the genetic trade-off relationships between traits should be taken into account in mass-rearing programs.     

Rhythmic components of photoperiodic time measurement in the pitcher-plant mosquito, Wyeomyia smithii

Photoperiodic time measurement regulating larval diapause in the pitcher-plant mosquito, Wyeomyia smithii, varies in a close relationship with latitude. The critical photoperiod mediating the maintenance and termination of diapause is positively correlated with latitude (r ² = 0.977) among six populations from southern (30–31° N), intermediate (40° N), and northern (46–49° N) latitudes in North America. The developmental response to unnaturally short and to unnaturally long photoperiods declines with increasing latitude, so that longer critical photoperiods are associated with a downward rather than a lateral shift in the photoperiodic response curve. Exotic light and dark cycles of varying period (T) with a short (10 h) photophase and a scotophase ranging from 14 (T = 24) to 62 (T = 72) h, reveal two geographic patterns: a decline in perturbability of the photoperiodic clock with increasing latitude, and no change with latitude in the 21-h period of rising and falling development with increasing T. These results show (1) that there is a rhythmic component to photoperiodic time measurement in W. smithii, (2) that the period of this rhythm is about 21 h in all populations, and (3) that more northern populations show decreasing responsiveness to photoperiod and increasing stability against perturbation by exotic period lengths (T > 24). Previous studies on W.␣smithii indicate that this single temperate species of a tropical and subtropical genus has evolved from south to north. We therefore conclude that the evolution of increasing critical photoperiod in W. smithii during its adaptive radiation into North America has more likely involved the amplitude and not the period of the underlying circadian pacemaker. Received: 22 July 1996 / Accepted: 30 September 1996     

Time, love and species

Physiological and behavioral phenomena of many animals are restricted to certain times of the day. Many organisms show daily rhythms in their mating. The daily fluctuation in mating activity of a few insects is controlled by an endogenous clock. The fruitfly, Drosophila, is the most suitable material to characterize the genetic basis of circadian rhythms of mating because some mutants with defective core oscillator mechanism, feedback loops, have been isolated. D. melanogaster wild-type display a robust circadian rhythm in the mating activity, and the rhythms are abolished in period or timeless null mutant flies (per(01) and tim(01)), the rhythms are generated by females but not males. Disconnected (disco) mutants which have a severe defect in the optic lobe and are missing lateral neurons show arrhythmicity in mating activities. Thus, the lateral neurons seem to be essential for the circadian rhythm in mating activity of Drosophila. Furthermore, an anti-phasic relation in circadian rhythms of the mating activity was detected between D. melanogaster and their sibling species D. simulans. The Queensland fruit flies or wild gypsy moth also show species-specific mating rhythm, suggesting that species-specific circadian rhythms in mating activity of insect appear to cause a reproductive isolation.     

An inter‐oscillator mechanism modulating circadian clock period in paramecium populations

Abstract

In populations of the ciliate protozoan, Paramecium multimicronucleatum, the circadian‐clock‐con‐trolled mating reaction expressed by a limited number of cells among them feeds back to contribute to coherence of their circadian rhythms of motility and mating reaction. This eventually causes a decrease in the period of the rhythms from the entrained 24h period to a steady‐state period of about 22h, with the rate of decrease depending on the strength of the mating reaction. These results suggest that the interaction among oscillators may be one of the factors which modulate the period of a circadian clock composed of nearly identical oscillators. The clock‐controlled mating reaction provides a promising inter‐oscillator pathway for obtaining more insight into the mechanism of modulation of the period of such circadian clocks through inter‐oscillator interaction.     

Independence of genetic variation between circadian rhythm and development time in the seed beetle, Callosobruchus chinensis

A positive genetic correlation between periods of circadian rhythm and developmental time supports the hypothesis that circadian clocks are implicated in the timing of development. Empirical evidence for this genetic correlation in insects has been documented in two fly species. In contrast, here we show that there is no evidence of genetic correlation between circadian rhythm and development time in the adzuki bean beetle, Callosobruchus chinensis. This species has variation that is explained by a major gene in the expression and period length of circadian rhythm between strains. In this study, we found genetic variation in development time between the strains. The development time was not covaried with either the incidence or the period length of circadian rhythm among the strains. Crosses between strains suggest that development time is controlled by a polygene. In the F₂ individuals from the crosses, the circadian rhythm is attributable to allelic variation in the major gene. Across the F₂ individuals, development time was not correlated with either the expression or the period length of circadian rhythm. Thus, we found no effects of major genes responsible for variation in the circadian rhythm on development time in C. chinensis. Our findings collectively give no support to the hypothesis that the circadian clock is involved in the regulation of development time in this species.     

Assortative mating,sexual selection,and their consequences for gene flow in Littorina

When divergent populations are connected by gene flow, the establishment of complete reproductive isolation usually requires the joint action of multiple barrier effects. One example where multiple barrier effects are coupled consists of a single trait that is under divergent natural selection and also mediates assortative mating. Such multiple-effect traits can strongly reduce gene flow. However, there are few cases where patterns of assortative mating have been described quantitatively and their impact on gene flow has been determined. Two ecotypes of the coastal marine snail, Littorina saxatilis, occur in North Atlantic rocky-shore habitats dominated by either crab predation or wave action. There is evidence for divergent natural selection acting on size, and size-assortative mating has previously been documented. Here, we analyze the mating pattern in L. saxatilis with respect to size in intensively sampled transects across boundaries between the habitats. We show that the mating pattern is mostly conserved between ecotypes and that it generates both assortment and directional sexual selection for small male size. Using simulations, we show that the mating pattern can contribute to reproductive isolation between ecotypes but the barrier to gene flow is likely strengthened more by sexual selection than by assortment.     

Patterns of reproductive isolation within and between two Lygaeus species characterized by sexual conflicts over mating

Theory suggests that, under some circumstances, sexual conflict over mating can lead to divergent sexually antagonistic coevolution among populations for traits associated with mating, and that this can promote reproductive isolation and hence speciation. However, sexual conflict over mating may also select for traits (e.g. male willingness to mate) that enhance gene flow between populations, limiting population divergence. In the present study, we compare pre‐ and post‐mating isolation within and between two species characterized by male–female conflict over mating rate. We quantify sexual isolation among five populations of the seed bug Lygaeus equestris collected from Italy and Sweden, and two replicates of a population of the sister‐species Lygaeus simulans, also collected from Italy. We find no evidence of reproductive isolation amongst populations of L. equestris, suggesting that sexual conflict over mating has not led to population divergence in relevant mating traits in L. equestris. However, there was strong asymmetric pre‐mating isolation between L. equestris and L. simulans: male L. simulans were able to mate successfully with female L. equestris, whereas male L. equestris were largely unable to mate with female L. simulans. We found little evidence for strong post‐mating isolation between the two species, however, with hybrid F₂ offspring being produced. Our results suggest that sexual conflict over mating has not led to population divergence, and indeed perhaps supports the contrary theoretical prediction that male willingness to mate may retard speciation by promoting gene flow.     

Entrainment of eclosion and preliminary ontogeny of circadian clock gene expression in the flesh fly,Sarcophaga crassipalpis

Timing of circadian activities is controlled by rhythmic expression of clock genes in pacemaker neurons in the insect brain. Circadian behavior and clock gene expression can entrain to both thermoperiod and photoperiod but the availability of such cues, the organization of the brain, and the need for circadian behavior change dramatically during the course of insect metamorphosis. We asked whether photoperiod or thermoperiod entrains the clock during pupal and pharate adult stages by exposing flies to different combinations of thermoperiod and photoperiod and observing the effect on the timing of adult eclosion. This study used qRT-PCR to examine how entrainment and expression of circadian clock genes change during the course of development in the flesh fly, Sarcophaga crassipalpis. Thermoperiod entrains expression of period and controls the timing of adult eclosion, suggesting that the clock gene period may be upstream of the eclosion pathway. Rhythmic clock gene expression is evident in larvae, appears to cease during the early pharate adult stage, and resumes again by the time of adult eclosion. Our results indicate that both patterns of clock gene expression and the cues to which the clock entrains are dynamic and respond to different environmental signals at different developmental stages in S. crassipalpis.     

The Role of Sexual Selection and Conflict in Mediating Among-Population Variation in Mating Strategies and Sexually Dimorphic Traits in Sepsis punctum

The black scavenger fly Sepsis punctum exhibits striking among-population variation in the direction and magnitude of sexual size dimorphism, modification to the male forelimb and pre-copulatory behaviour. In some populations, male-biased sexual size dimorphism is observed; in other, less dimorphic, populations males court prior to mating. Such variation in reproductive traits is of interest to evolutionary biologists because it has the potential to limit gene flow among populations, contributing to speciation. Here, we investigate whether large male body size and modified forefemur are associated with higher male mating success within populations, whether these traits are associated with higher mating success among populations, and if these traits carry viability costs that could constrain their response to sexual selection. Flies from five distinct populations were reared at high or low food, generating high and low quality males. The expression of body size, forelimb morphology and courtship rate were each greater at high food, but high food males experienced higher mating success or reduced latency to first copulation in only one of the populations. Among populations, overall mating success increased with the degree of male-bias in overall body size and forelimb modification, suggesting that these traits have evolved as a means of increasing male mating rate. The increased mating success observed in large-male populations raises the question of why variation in magnitude of dimorphism persists among populations. One reason may be that costs of producing a large size constrain the evolution of ever-larger males. We found no evidence that juvenile mortality under food stress was greater for large-male populations, but development time was considerably longer and may represent an important constraint in an ephemeral and competitive growth environment.     

Divergent selection and the evolution of signal traits and mating preferences

Mating preferences are common in natural populations, and their divergence among populations is considered an important source of reproductive isolation during speciation. Although mechanisms for the divergence of mating preferences have received substantial theoretical treatment, complementary experimental tests are lacking. We conducted a laboratory evolution experiment, using the fruit fly Drosophila serrata, to explore the role of divergent selection between environments in the evolution of female mating preferences. Replicate populations of D. serrata were derived from a common ancestor and propagated in one of three resource environments: two novel environments and the ancestral laboratory environment. Adaptation to both novel environments involved changes in cuticular hydrocarbons, traits that predict mating success in these populations. Furthermore, female mating preferences for these cuticular hydrocarbons also diverged among populations. A component of this divergence occurred among treatment environments, accounting for at least 17.4% of the among-population divergence in linear mating preferences and 17.2% of the among-population divergence in nonlinear mating preferences. The divergence of mating preferences in correlation with environment is consistent with the classic by-product model of speciation in which premating isolation evolves as a side effect of divergent selection adapting populations to their different environments.     

Circadian rhythm and time of mating in Bactrocera cucurbitae (Diptera: Tephritidae) selected for age at reproduction

Chrono-biological traits were changed by selecting for life-history traits via a genetic linkage controlling both time-related behavioural and life-history traits. Behavioural traits were compared between lines selected for young (Y-lines) and old (O-lines) age at reproduction in the melon fly, Bactrocera cucurbitae (Coquillett). Adults from O-lines, which survive longer than flies from Y-lines, mated later in the day and had a longer period of circadian rhythm in the locomotor activity than those from Y-lines. Flies from F(1) reciprocal crosses had an intermediate time of mating and periods of circadian rhythm between that of the parents, indicating a genetic basis to these traits. The presence of these behavioural differences across the selection lines indicates that chrono-biological traits exhibit correlated responses to selection on age at reproduction. The correlated responses in the behavioural traits to selection for life-history traits are discussed from two points of view: pleiotropy and inadvertent selection.     

Evolution of mating behavior between two populations adapting to common environmental conditions

Populations from the same species may be differentiated across contrasting environments, potentially affecting reproductive isolation among them. When such populations meet in a novel common environment, this isolation may be modified by biotic or abiotic factors. Curiously, the latter have been overlooked. We filled this gap by performing experimental evolution of three replicates of two populations of Drosophila subobscura adapting to a common laboratorial environment, and simulated encounters at three time points during this process. Previous studies showed that these populations were highly differentiated for several life‐history traits and chromosomal inversions. First, we show initial differentiation for some mating traits, such as assortative mating and male mating rate, but not others (e.g., female mating latency). Mating frequency increased during experimental evolution in both sets of populations. The assortative mating found in one population remained constant throughout the adaptation process, while disassortative mating of the other population diminished across generations. Additionally, differences in male mating rate were sustained across generations. This study shows that mating behavior evolves rapidly in response to adaptation to a common abiotic environment, although with a complex pattern that does not correspond to the quick convergence seen for life‐history traits.     

Flies, clocks and evolution.

The negative feedback model for gene regulation of the circadian mechanism is described for the fruitfly, Drosophila melanogaster. The conservation of function of clock molecules is illustrated by comparison with the mammalian circadian system, and the apparent swapping of roles between various canonical clock gene components is highlighted. The role of clock gene duplications and divergence of function is introduced via the timeless gene. The impressive similarities in clock gene regulation between flies and mammals could suggest that variation between more closely related species within insects might be minimal. However, this is not borne out because the expression of clock molecules in the brain of the giant silk moth, Antheraea pernyi, is not easy to reconcile with the negative feedback roles of the period and timeless genes. Variation in clock gene sequences between and within fly species is examined and the role of co-evolution between and within clock molecules is described, particularly with reference to adaptive functions of the circadian phenotype.     

Sexual isolation promotes divergence between parapatric lake and stream stickleback

Speciation can be initiated by adaptive divergence between populations in ecologically different habitats, but how sexually based reproductive barriers contribute to this process is less well understood. We here test for sexual isolation between ecotypes of threespine stickleback fish residing in adjacent lake and stream habitats in the Lake Constance basin, Central Europe. Mating trials exposing females to pairings of territorial lake and stream males in outdoor mesocosms allowing for natural reproductive behaviour reveal that mating occurs preferentially between partners of the same ecotype. Compared to random mating, this sexual barrier reduces gene flow between the ecotypes by some 36%. This relatively modest strength of sexual isolation is surprising because comparing the males between the two ecotypes shows striking differentiation in traits generally considered relevant to reproductive behaviour (body size, breeding coloration, nest size). Analysing size differences among the individuals in the mating trials further indicates that assortative mating is not related to ecotype differences in body size. Overall, we demonstrate that sexually based reproductive isolation promotes divergence in lake–stream stickleback along with other known reproductive barriers, but we also caution against inferring strong sexual isolation from the observation of strong population divergence in sexually relevant traits.