Flash signal evolution in Photinus fireflies: Character displacement and signal exploitation in a visual communication system

Animal communication is an intriguing topic in evolutionary biology. In this comprehensive study of visual signal evolution, we used a phylogenetic approach to study the evolution of the flash communication system of North American fireflies. The North American firefly genus Photinus contains 35 described species with simple ON–OFF visual signals, and information on habitat types, sympatric congeners, and predators. This makes them an ideal study system to test hypotheses on the evolution of male and female visual signal traits. Our analysis of 34 Photinus species suggests two temporal pattern generators: one for flash duration and one for flash intervals. Reproductive character displacement was a main factor for signal divergence in male flash duration among sympatric Photinus species. Male flash pattern intervals (i.e., the duration of the dark periods between signals) were positively correlated with the number of sympatric Photuris fireflies, which include predators of Photinus. Females of different Photinus species differ in their response preferences to male traits. As in other communication systems, firefly male sexual signals seem to be a compromise between optimizing mating success (sexual selection) and minimizing predation risk (natural selection). An integrative model for Photinus signal evolution is proposed.     

Spontaneous flash communication of females in an Asian firefly

Fireflies are well known for the use of bioluminescence for sexual communication. In species using flash signals for pair formation, species and sexual identity are conferred by flash timing parameters such as flash duration, flash interval, flash number, and response delay. In dialog fireflies in North America, the male is the advertiser and the female is the responder. In these species, the male flash signal parameter varies depending on species, but the female flash signal parameter is limited only to response delay. However, in fireflies other than dialog fireflies, sexual flash communication is not well studied. Although many female-advertisement-like fireflies are reported, we have no confirmed case of sexual communication in a female-advertisement species. Here, we report the sexual flash communication of an Asian firefly, Luciola (Hotaria) parvula, in which the female flashes spontaneously. By using an electronic firefly, we confirm experimentally that males are specifically attracted to flashes with a female-specific flash duration. This is the first experimental report of sexual communication of a female advertiser in firefly communication. In this species, females call males usually with spontaneous flashes unlike dialog fireflies.     

Variation in flash speed of Japanese firefly,Luciola cruciata (Coleoptera: Lampyridae), identifies distinct southern “quick‐flash” population on Goto Islands,Japan

Luciola cruciata, a bioluminescent firefly endemic to Japan, communicates with species‐specific flashing patterns. Previous studies reported that flying males possess three distinct flashing frequencies: slow (4 s), intermediate (3 s) and fast (2 s). Our study focused on geographical variations in flashing patterns in the northwestern part of the Kyushu region of southern Japan (including its isolated islands) and their relationship to the mitochondrial COII region of firefly DNA. We found that “quick‐flash (1 s)” fireflies occur in the Goto Islands, at the western tip of Kyushu, and that, although they possess a unique haplotype, they do not differ much genetically from “fast‐flash” fireflies inhabiting mainland Kyushu. In contrast, although fireflies inhabiting the Tsushima and Iki Islands also possess a unique haplotype, their flashing patterns were the same (fast‐flash) as fireflies inhabiting mainland Kyushu. We determined that flash patterns of L. cruciata in Japan should be classified according to four flashing patterns: slow, intermediate, fast and quick.     

Firefly Courtship: Behavioral and Morphological Predictors of Male Mating Success in Photinus greeni

Differences in male mating success can generate selection on male morphological traits and courtship behaviors involved in male–male competition or female mate choice. In Photinus fireflies (Coleoptera: Lampyridae), courtship is based on bioluminescent flash signals produced by both sexes. We conducted field observations of Photinus greeni fireflies engaged in competitive courtships, in which females are able to simultaneously assess several males, to identify male morphological traits and courtship behaviors that might predict male mating success. Male morphological traits did not differ between males that successfully mated compared with unsuccessful males (dialoging males that did not mate). However, courtship behavioral interactions differed: successful males tended to have higher flash pattern rates (number of flash patterns per minute), and their courtship flashes were more likely to be answered by females. We also examined how the risk of predation by Photuris fireflies altered courtship behavior of their Photinus prey. When predatory Photuris fireflies were present, P. greeni females were less likely to mate, and showed decreased flash responses to most males. However, P. greeni males that did successfully mate in spite of Photuris presence were males that maintained high flash pattern rates that elicited female responses. These results suggest that both female mate choice and Photuris predation exert strong selective pressures on the evolution of courtship signals in Photinus fireflies.     

The occurrence of synchrony in the North American fireflyPhotinus carolinus (Coleoptera: Lampyridae)

Synchronous flashing has been described for some Southeast Asian fireflies but has rarely been reported in North American fireflies. Our field observations indicated thatPhotinus carolinus flash synchronously. The flash pattern of individualP. carolinus was characterized by a burst of five to eight flashes over a period of approximately 4 s. These flash bursts were repeated about every 12 s. Groups of fireflies comprised individuals exhibiting this species-specific flash pattern. Remarkably, members of the group flashed synchronously: Flash bursts started and stopped at the same time and the flashes among individuals occurred at the same time as well. We used low-light level videography to examine this behavior in caged groups of (3 or 10) fireflies for synchronic flashing. The occurrence of concurrent rhythmic group flashing satisfies the criteria for synchrony as defined by Buck (1988). The intermittent nature of the bursts of flashes shown byP. carolinus makes this a discontinuous synchrony. The mechanisms underlying discontinuous synchrony are not known.     

The ecological impact of an introduced population on a native population in the firefly Luciola cruciata (Coleoptera: Lampyridae)

In Japan, Tatsuno Town has been famous for many Luciola cruciata fireflies emerging every summer at least since the 1920s. However, in the 1960s, L. cruciata fireflies were intentionally introduced from the Lake Biwa area into Matsuo-kyo, the most famous habitat of fireflies in that town. In this study, I examined ambient temperatures and flash rates of L. cruciata at four sites including Matsuo-kyo in the Tatsuno area and two sites in the Lake Biwa area. The linear regression of flash rates on temperatures indicated that the Matsuo-kyo population was distinct from the other three populations native to the Tatsuno area, but similar to the two populations native to the Lake Biwa area in terms of flash rates. These results were also supported by a recent molecular biological study, suggesting that the introduced fireflies had a strong ecological impact on the native ones at Matsuo-kyo. The present study emphasizes that we should not transport and release L. cruciata fireflies without careful consideration.     

Biochemical systematics of Japanese fireflies of the subfamily Luciolinae and their flash communication systems

Japanese fireflies of the subfamily Luciolinae are biochemically analyzed using 13 allozymes, and the phylogenetic relationships obtained from this analysis are compared with their flash communication systems. As a result, the Japanese Luciolinae can be divided into three groups.Hotaria parvula andH. tsushimana together withLuciola yayeyamana andL. kuroiwae from the first group, and they use the same communication system.L. lateralis, Curtos okinawana, andC. costipennis make up the second group, and their communication systems are also the same.L. cruciata makes up the last one, and its communication system is different from the other fireflies of Luciolinae. Therefore, their taxonomical arrangement and communication systems are not congruent. However, the genetic similarity deduced by allozymic analysis of the members of the Japanese Luciolinae is highly consistent with their flash communication systems.     

Assessing Condition-dependence of Male Flash Signals in <Emphasis Type="Italic">Photinus</Emphasis> Fireflies

Females often show a preference for exaggerated male sexual traits or courtship behaviors. Such preferences can benefit females if trait expression is correlated with male genetic quality or phenotypic condition. Previous studies of several Photinus fireflies have revealed considerable intraspecific variation in the bioluminescent courtship signals emitted by males, and have also demonstrated that females prefer more conspicuous male signals. Thus, females might gain information about male phenotypic quality if courtship signals reflect male condition. We examined possible condition-dependence of Photinus male courtship signals using two complementary approaches. First we experimentally manipulated male mating status, which is expected to affect male condition by depleting resources required for nuptial gift formation, and looked at how individual male flash signals changed with mating status and over time. We used an additional approach to assess condition-dependence by examining whether a relationship exists between flash signal parameters and measures of male condition and body shape. We found that the pulse rate of P. greeni courtship signals was not altered by male mating status or age, and that the pulse duration of P. ignitus signals was also not affected by male mating status. In P. pyralis fireflies, males showed a non-significant trend toward reduced signal pulse duration with age. When we examined the relationship between male flash signals and condition measures, we found no effect of male condition or body shape on courtship signals in P. greeni or P. ignitus; in P. pyralis, males with wider body shapes produced longer duration flash signals. On the other hand, we found no evidence in P. pyralis that condition predicted flash duration. Taken together, these results indicate that Photinus males’ flash signals do not reflect adult male condition, and suggest that females are unlikely to use courtship signals as an indicator of male phenotypic quality.     

Flash Precision at the Start of Synchrony in Photuris frontalis

Synchronous flashing occurs in certain species of SoutheastAsian and North American fireflies. Most Southeast Asian synchronyinvolves stationary congregating fireflies, but North Americansynchrony occurs in flying fireflies that do not congregate.Southeast Asian synchrony is usually continuous, but North Americansynchrony is interrupted. Photuris frontalis, the only memberof the North American genus Photuris to synchronize, shows anintermittent synchrony. This involves synchronization and repeatedre-synchronizations while in flight. The precision that occursat the start of synchrony was studied in Ph. frontalis usingcaged fireflies and photometry. Barrier experiments (using twofireflies) or flash entrainment experiments (using one LED andone firefly) were performed to measure the temporal precisionof the first entrained flash. In both cases, the first entrainedflash was close to unison synchrony (phase = 1.0) and showedlittle variability. The behavioral implications of the abilityto synchronize with the first entrained flash are not known,but it might facilitate male-male interactions during brief,transient encounters such as maintaining distance between closelyflying males in search of females.     

Pyrazine emission by a tropical firefly: An example of chemical aposematism?

Although famous for photic courtship displays, fireflies (Coleoptera: Lampyridae) are also notable for emitting strong odors when molested. The identity of volatile emissions and their possible role, along with photic signals, as aposematic warnings of unpalatability have been little explored, especially in tropical species. Pursuant to the observation that the widespread Neotropical fireflies, Photuris trivittata and Bicellonycha amoena, emit pungent odors, glows, and flashes when handled, we investigated their cuticular and headspace chemistry. Gas chromatography–mass spectrometry analyses revealed that both fireflies have species‐specific cuticular hydrocarbon profiles. Photuris trivittata headspace was dominated by 2‐methoxy‐3‐(1‐methylpropyl) pyrazine (hereafter, pyrazine), on the order of 1.59 ng/individual and a suite of sesquiterpenes, while B. amoena emitted 3‐methoxy‐2‐butenoic acid methyl ester and a few ketones. This is the first report of such compounds in fireflies. We investigated the role of pyrazine in P. trivittata's interactions with potential predators: sympatric ants, toads, and bats. Solvent‐washed P. trivittata painted with pyrazine incurred lower ant predation than did their solvent‐washed counterparts. Pyrazine significantly repelled ants at baits in concentrations as low as 9.8 × 10^?4 ng/μl. The toad, Rhinella marina, readily accepted intact fireflies, pyrazine‐coated and uncoated mealworms. Both Myotis nigricans and Molossus molossus bats rejected fireflies, but accepted both pyrazine‐coated and uncoated mealworms. While pyrazine repels ants, its role as an aposematic signal warning other potential predators of firefly distastefulness requires further investigation. Our results underscore the idea that multiple enemies exert conflicting selection on firefly defenses.     

Male courtship signals and female signal assessment in Photinus greeni fireflies

The evolutionary dynamic of courtship signaling systems is drivenby the interaction between male trait distributions and femalepreferences. This interaction is complex because females maychoose mates based on multiple components of male signals, andfemale preference functions may vary depending on mate availability,female reproductive state, and environmental conditions. InPhotinus fireflies (Coleoptera: Lampyridae), flying males emitbioluminescent flash signals to locate sedentary females, whichreply selectively to attractive male flash signals with theirown response flash. In this study, we first examined temporalvariation in the paired-pulse flash patterns produced by Photinusgreeni males in the field and found significant among-male variation(70% of total variation) in interpulse intervals (IPIs). Therewas no significant relationship between male IPI and spermatophoresize, suggesting that P. greeni male courtship signals do notprovide females with reliable indicators of male material resources.In laboratory playback experiments, we presented P. greeni femaleswith simulated flash signals to assess how IPI and pulse durationindependently affected the likelihood of female flash response.We also examined the effects of female body mass and time duringthe mating season on female preference functions, hypothesizingthat females would be less discriminating when they were heavier(more fecund) and when mate availability declined. We foundthat P. greeni females discriminated among signals within theirspecies' range based primarily on flash pattern IPI. Neitherthe time during the mating season nor female weight alteredfemale preference functions for IPI, although season did influencefemale response to pulse duration. These results reveal thatP. greeni females discriminate among conspecific males basedprimarily on male IPIs, the same signal character previouslyshown to be important for firefly species recognition. Fieldplayback experiments indicated that female responsiveness peakednear the average IPI given by males at different ambient temperatures,suggesting that fireflies exhibit temperature coupling similarto that seen in many acoustically signaling animals.     

Variation in the flash pattern of the firefly,Photuris versicolor quadrifulgens (Coleoptera: Lampyridae)

We sampled a population of signalingPhoturis versicolor quadrifulgens fireflies to quantify the variation in flash patterns emitted by males. Males produced five distinct flash patterns during their mate-searching flights. Four of the patterns consisted of two to five equal-intensity pulses and the fifth pattern type was a flicker, a group of rapid modulations in intensity. We found that the proportions of each pattern remained relatively constant from night to night throughout the season. The different flash patterns produced varied significantly with time of night; patterns having fewer pulses occurred earlier in the evening. Local density, an estimate of competition, did not significantly correlate with flash pattern type. On consecutive emissions, individuals changed their flash types with a mean probability of 0.12 (over all males), and they usually switched between patterns differing by a single pulse (from a two- to a three-pulse pattern, from a three- to a two-pulse pattern, etc.). The nightly temporal changes in flash patterns may be related to tradeoffs between female availability and energetic costs of signaling or the changes may be related to increased predation risk from visual predators. Photuris versicolor quadrifulgens was originally described by Barber (1951) as a subspecies ofP. versicolor. The genusPhoturis is currently under revision by Dr. James E. Lloyd, and this firefly will be given species status.     

SuperSegger: robust image segmentation,analysis and lineage tracking of bacterial cells

Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame‐to‐frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB‐based image processing package well‐suited to quantitative analysis of high‐throughput live‐cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine‐learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame‐to‐frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell‐cycle dynamics in bacteria as well as cell‐contact mediated phenomena. This package has a range of built‐in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution.     

Flash signals, nuptial gifts and female preference in photinus fireflies

The evolution of male courtship signals such as the bioluminescentflashes of fireflies may be shaped, at least in part, by femalepreference for particular characteristics of the male signal.These female preferences for male courtship signals may ariseas a result of the benefits of choosing males with particulartraits. One possible benefit of mate choice occurs if femalescan use male courtship signals as an honest indicator of malenutritional contributions at mating, nuptial gifts. This paperreviews female preference for male flash characteristics inPhotinus fireflies (Coleoptera: Lampyridae), and the potentialfor females to use male flash characteristics to predict nuptialgift quality. In Photinus firefly species with single pulseflashes females preferentially respond to flashes of greaterintensity and duration. Male Photinus provide a nuptial giftto females at mating in the form of a spermatophore and flashduration serves as a good predictor of spermatophore mass formales collected early in the season. However, Photinus firefliesdo not feed as adults, so spermatophore mass decreases withsubsequent matings. In response, nutrient-limited females maystop preferentially responding to longer duration flashes, increasingtheir overall responsiveness later in the mating season as theyforage for spermatophores. Therefore, the evolution of malecourtship signals in Photinus fireflies is the product not onlyof female preference for male flash characteristics, but alsothe costs and benefits of female choice that shape these preferences.     

A New Type of Synchronized Flashing in a North American Firefly

Continuous synchronic flashing occurs in stationary and flying male congregations of members of some firefly species in Southeast Asia. In the present paper, low-light videography and photometry was used to demonstrate that synchrony occurs in the North American genus Photuris. We found that the Georgia coastal plain firefly Ph. frontalis flashed synchronically. From a distance, the synchronic flashes of a population of flying Ph. frontalis appeared to occur in a continuous synchrony. However, when pairs of males were viewed, it was difficult to verify that their flashing was continuously synchronic. In the laboratory, caged fireflies flashed synchronically, stopped, and then flashed synchronically again. To study this flash behavior, recordings of individual and group flashing were analyzed statistically to validate the conclusions about rhythm and synchrony. Although the mass synchrony appeared continuous, the individual flying males switch on and off, coming in again on the beat. The synchrony in Ph. frontalis is common and pervasive rather than rare and sporadic, as shown by other North American fireflies. The precision of the frontalis synchrony approaches that of Southeast Asian fireflies. The intermittent synchrony of this firefly reinforces the evidence that there is a diversity of synchronies in fireflies.     

Flight Studies on Photic Communication by the Firefly Photinus pyralis

Flash communication by the firefly Photinus pyralis was studiedin a stationary, simulated flight apparatus in which an individualof either sex could be "flown" and its flashing behavior andflight orientation recorded in response to photic stimulation.Males made long "flights" showing many of the characteristicsof their natural, female-seeking patrol flights. Males orientedtheir flight vectors towards light emitting diode (LED) flashesthat mimicked the responses of females to their patrol flashes.Females flew and responded to male-emulating LED flashes, makinga previously unknown early response followed by the typical2 sec delayed response characteristic of the dialoging perchedfemale, including abdominal aiming of the flash. Pairs consistingof males, in tethered flight, and females, perched, were runin an integrating sphere photometer, permitting the first determinationsof flash intensities of both sexes during courtship dialog.The implications of this work on thought about evolution ofphotic behavior in fireflies are considered.     

Flash behavior of femalePhoturis versicolor fireflies (Coleoptera: Lampyridae) in simulated courtship and predatory dialogues

FemalePhoturis versicolor fireflies attempt to capture males by responding to heterospecific flash patterns. A mating-dependent switch occurs which affects response timing and frequency of female flashes. We examined the switch using females of known age, mating status, and flash experience to assess how accurate mimicry is, what factors influence it, and what mechanism produces it. Presentations of simulated male flash patterns before and after mating revealed elements of an entrainment mechanism controlling female responsiveness. Unmated females preferentially answered conspecific patterns with variable latencies, averaging 1 s. Mating induced changes in both response frequency and response latency: Females answered heterospecific patterns more frequently, and latencies elicited by conspecific patterns shifted away from the unmated range. Heterogeneity in mean and variance of response latency among individuals indicates that females do not share a discrete reply to a given pattern. Little correspondence exists between latencies of sympatric species andP. versicolor females, suggesting that the flash response mechanism produces entriainment to any rhythmic pattern, not a one-to-one matching between prey and predator latencies. Different selective scenarios underlie strict mimicry versus entrainment mimicry.     

Abdomen Posture and Flash Gesture Direction in a Female Synchronic Firefly <Emphasis Type="Italic">Photinus carolinus</Emphasis> (Green) (Coleoptera: Lampyridae)

North American Photinus fireflies use bioluminescent flashes to communicate an individual’s species and sex, and to attract potential mates. A female firefly responds to a male firefly’s courtship flash with her own species-specific flash. We used a photic stimulator to produce male-like species-specific P. carolinus LED courtship flashes. These evoked species-specific response flashes from a female. The female’s flashes were preceded by a flash gesture comprising a sequence of abdominal postural adjustments (pitch, roll, and yaw). These gestures changed her lantern’s orientation which, at rest, was downward towards the substrate. Our results demonstrate that these gestures mediate a lateralization of the female’s response flashes towards the direction of the stimulating LED. That is, she directs her response to the left of midline when stimuli are presented from her left, and similarly, she directs her response to the right of midline when stimuli are presented from her right. The directional aspect of the flash gesture adds a new perspective to the complexity of the behaviors associated with flash communication in fireflies. Lateralization of the flash gesture suggests that the female’s visual system processes information about the location of male’s flashes as well as their temporal pattern.     

Biased dispersal of Metrioptera bicolor,a wing dimorphic bush‐cricket

In the highly fragmented landscape of central Europe, dispersal is of particular importance as it determines the long‐term survival of animal populations. Dispersal not only secures the recolonization of patches where populations went extinct, it may also rescue small populations and thus prevent local extinction events. As dispersal involves different individual fitness costs, the decision to disperse should not be random but context‐dependent and often will be biased toward a certain group of individuals (e.g., sex‐ and wing morph‐biased dispersal). Although biased dispersal has far‐reaching consequences for animal populations, immediate studies of sex‐ and wing morph‐biased dispersal in orthopterans are very rare. Here, we used a combined approach of morphological and genetic analyses to investigate biased dispersal of Metrioptera bicolor, a wing dimorphic bush‐cricket. Our results clearly show wing morph‐biased dispersal for both sexes of M. bicolor. In addition, we found sex‐biased dispersal for macropterous individuals, but not for micropters. Both, morphological and genetic data, favor macropterous males as dispersal unit of this bush‐cricket species. To get an idea of the flight ability of M. bicolor, we compared our morphological data with that of Locusta migratoria and Schistocerca gregaria, which are very good flyers. Based on our morphological data, we suggest a good flight ability for macropters of M. bicolor, although flying individuals of this species are seldom observed.