Allometry of a sexual trait in relation to diet experience and alternative mating tactics in two rubyspot damselflies (Calopterygidae: Hetaerina)

Several arguments have been put forward to explain how sexual selection drives the evolution of sexual trait allometry, especially hyperallometry. The ‘positive allometry theory’ suggests that hyperallometry is a rule in all‐secondary sexual traits, whereas the ‘display hypothesis’ suggests that only males in good condition will exhibit hyperallometric sexual display traits. In the present study, we investigated: (1) the condition‐dependence nature (by using two diet treatments that varied in the amount of food provided to the larvae) of a sexually selected trait (wing pigmentation; WP) in recently‐emerged adults of the American rubyspot damselfly, Hetaerina americana, and (2) the scaling relationship between WP and body size (wing and body length) in the rubyspot damselflies H. americana and Hetaerina vulnerata, according to alternative reproductive tactics (ARTs; territorial and nonterritorial males). First, we found support that indicated that diet positively affected WP length, although there was no significant WP allometric pattern in relation to diet regimes. Second, WP was hyperallometric in both Hetaerina species. WP size was similar between ARTs and, in H. americana (but not H. vulnerata), nonterritorial males showed steeper slopes than territorial males when wing length was used. The results obtained support the notion that sexual traits are hyperallometric, although there is no clear pattern in relation to ARTs. © 2013 The Linnean Society of London     

Character displacement in the fighting colours of Hetaerina damselflies

Aggression between species is a seldom-considered but potentially widespread mechanism of character displacement in secondary sexual characters. Based on previous research showing that similarity in wing coloration directly influences interspecific territorial aggression in Hetaerina damselflies, we predicted that wing coloration would show a pattern of character displacement (divergence in sympatry). A geographical survey of four Hetaerina damselfly species in Mexico and Texas showed evidence for character displacement in both species pairs that regularly occurs sympatrically. Hetaerina titia, a species that typically has large black wing spots and small red wing spots, shifted to having even larger black spots and smaller red wing spots at sites where a congener with large red wing spots is numerically dominant (Hetaerina americana or Hetaerina occisa). Hetaerina americana showed the reverse pattern, shifting towards larger red wing spots where H. titia is numerically dominant. This pattern is consistent with the process of agonistic character displacement, but the ontogenetic basis of the shift remains to be demonstrated.     

Survival cost of an intrasexually selected ornament in a damselfly

Ornaments could evolve as honest indicators of fighting ability, provided they have costs that make deceptive signalling unprofitable. I tested for such costs by manipulating the size of the intrasexually selected wing spots of male rubyspot damselflies (Hetaerina americana) and monitoring survival in the field. Males with enlarged spots had higher mortality rates than both unmanipulated and sham-manipulated controls. Natural wing spot size correlated positively with longevity, which suggests that higher quality males develop larger spots.     

INTRASEXUAL COMPETITION ALONE FAVORS A SEXUALLY DIMORPHIC ORNAMENT IN THE RUBYSPOT DAMSELFLY HETAERINA AMERICANA

I studied the sex-limited red spots on the wings of male rubyspot damselflies (Hetaerina americana) in relation to territoriality and fitness in the wild. Both observational and experimental (wing spot manipulation) studies indicated that wing spots were selected through competition among males for mating territories, not through female choice or direct competition for females. Males with naturally or artificially large wing spots were more successful at holding territories and consequently mated at higher rates than males with relatively small wing spots. In contrast, sexual selection on male body size appeared to operate among nonterritorial males at the clasping stage of the mating sequence, perhaps because larger males were better at clasping females forcibly. Of four models proposed to explain the evolution of ornaments through territory competition, only the agonistic handicap model makes predictions consistent with the results of this study.     

SEXUAL SELECTION AND SURVIVAL SELECTION ON WING COLORATION AND BODY SIZE IN THE RUBYSPOT DAMSELFLY HETAERINA AMERICANA

I review methodological problems that can lead to false evidence for selection on secondary sexual characters and present a study of selection in rubyspot damselflies (Hetaerina americana) that avoids these pitfalls. Male rubyspots have a large red spot on each wing that grows to a terminal size after sexual maturity. Selection gradient analyses revealed evidence for positive sexual and survival selection on both terminal wing spot size and body size. Phenotype manipulations confirmed that wing spot size was subject to direct sexual selection, but showed that the positive slope of survival on wing spot size was an indirect effect of selection on unmeasured traits. This study provides the strongest evidence yet for sexual selection on coloration in Odonata, but also provides clear examples of why phenotypic selection statistics must be calculated and interpreted cautiously.     

Physiological condition and wing pigmentation expression in a damselfly with seasonal polyphenism

Secondary sexual traits can be indicators of individual condition that may present seasonal polyphenism as a result of the differential costs of expression along the season. Wing spots in male damselflies of the Calopterygidae family are secondary sexual traits associated with intrasexual competition and mate choice. Hetaerina titia Drury is a calopterygid damselfly where males show red and black wing spots, contrasting with other species of the genus whose males only express a red wing spot. In the present study, we evaluate the seasonal variation of the expression of male's red and black wing spots and their allometric patterns. Additionally, we measure male condition in the form of proteins, lipids, soluble carbohydrates and glycogen in early and late seasons. Black wing spots present higher variation than red wing spots and males of the late season are more pigmented. Allometry is positive for wing red spot in the early season and for black spot in the late season. Males of the late season present a higher concentration of proteins, soluble carbohydrates and glycogen, although there is no variation in the lipid content. The results of the present study suggest that, in H. titia males, black pigmentation replaces the function of the red pigmentation to signal condition. Both traits, however, may be heavily affected by environmental situations (e.g. food availability).     

Is allometry of sexual traits adaptive? A field test with territorial damselflies

Recent studies have linked static allometry of sexual traits to selective advantages, in terms of sexual selection. An underlying, yet untested, assumption is that the allometry of sexual traits confers higher mating success and/or survival. Here, we investigated whether the allometry of two sexual traits is related to male mating success and survival in two species of damselflies: wing size in Paraphlebia zoe and the red‐pigmented wing spot in Hetaerina americana. We used large field‐based data sets of marked‐recaptured animals, in which we recorded male mating success and survival. Both sexual traits exhibited hyperallometric patterns; however, allometry was not linked to either mating success or survival. These results indicate that, at least during the period of sexual competition, allometry does not seem to be adaptive. Although our results may only apply to our damselfly study subjects (which nevertheless would require further tests in different seasons and/or study sites), our findings should encourage researchers to evaluate at least whether the assumed adaptiveness of sexual trait allometry holds for their study animals. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 327–334.     

Spatial and temporal population differences in male density and condition in the American rubyspot, Hetaerina americana (Insecta: Calopterygidae)

Increased resource availability should favor higher animal density. It may also affect sex ratio, the male condition, and mating competition over access to females, although the direction of these variables is not straightforward to predict. Using a non-experimental approach, we carried out preliminary research using the territorial American rubyspot (Hetaerina americana) by comparing two spatially separated populations and the same population in two different seasons (each comparison with varying population densities). We first compared the sex ratio by counting males (using two categories, territorial and non-territorial) and females; relative foraging time (as an indicator of how much feeding resources each site provides); wing spot size (a sexual ornament), body size and immune melanization response (these two variables were used to assess male condition); and fighting time and contest number (to assess competition). For the seasonal comparison we used a third population in which we only compared male spot size and two indicators of condition and immune response, phenoloxidase (PO) and nitric oxide (NO) activity. The high-density population had higher values of non-territorial males (but similar sex ratio), relative foraging time, contest time and number, wing spot (but similar body size) and melanization response than the low-density population. According to season, at high density, males had higher values of wing spots, PO, and NO. Our results suggest that in a population where animals have more dietary resources, males reach a better condition despite the competition being more intense.     

Resource defense polygyny by Hetaerina rosea Selys (Odonata: Calopterygidae): influence of age and wing pigmentation

Current evidence suggests that in Hetaerina damselflies males exhibit lek mating system. In this study, in order to answer if the same occurs in Hetaerina rosea Selys, we manipulated vegetation substrates used as territories and quantified the number of visiting females, males defending territories and fight intensity. We also examined whether body size and wing pigmentation are selectable traits in male-male competition, and if age affects male territorial behavior. Our results showed that males with larger pigmented areas won more contests, independently of body size. Old males changed from territoriality to sneaking strategy. Contrary to other Hetaerina species, males of H. rosea do not display lek behavior, but defend resources according to the resource defense polygyny strategy.     

Territorial damselflies do not show immunological priming in the wild

Adaptive immunity allows vertebrates to gain protection against repeated pathogenic infections. Analogous responses (priming) have been recently uncovered in invertebrates. However, whether such responses are widespread is not known. The present study investigated the presence of immunological priming in males of a species whose phylogenetic position places it in one of the less derived insect orders. It is hypothesized that the efficiency of such a response could be related to animal condition, as assessed by the expression of a sexually selected ornament. Hetaerina americana Fabricius (Odonata: Calopterygidae) males bear a conspicuous ornament (a red wing spot), which is evolutionarily maintained via male territorial competition. Using field‐collected animals, a group of males is challenged with bacteria before exposure to a higher dose of the same or a different bacteria, and survival is compared with that of infected males not previously challenged, as well as control groups. Gram‐positive and Gram‐negative bacteria are used. To explore how long priming may take to work, the second exposure is carried out either after 1 or 5 days. Red spot and body size are entered in the analysis as predictors of survival within and between groups. There is no difference in survival among groups, which suggests no priming effect. Overall, red spot and body size are not consistent in explaining survival.     

Condition dependence and trade-offs of sexual versus non-sexual traits in an insect

Sexual traits often communicate male condition and so are known to be highly condition-dependent. Thus, it is expected that, under restricted environments, sexual traits will be more heavily impacted than non-sexual traits, and so a negative covariation will be expected between sexual traits and non-sexual traits as only high-quality males will sustain the costs of producing both trait types. Such covariation will not necessarily appear in non-restricted environments. We tested these predictions using males of the American rubyspot, Hetaerina americana. First, fully mature males from different seasons were collected and their sexual [a wing red spot and body size (this corrected for body mass using residuals)], and condition-indicating, non-sexual (phenoloxidase and protein concentration) traits were measured. Second, larvae were reared under different food quantities and the same traits plus another non-sexual trait [pro-phenoloxidase (proPO)], were measured in recently emerged males. Contrary to expected, non-sexual traits showed larger expression variance than sexual traits. We found a significant covariation between body size and proPO for experimental males. Both rich and poor diet groups showed a negative slope for body size and proPO. This supposes a resource allocation trade-off between these two traits for recently emerged animals. On the other hand, the presumed signaling function between sexual traits, such as spot size, and physiological indicators of condition in this species, is not supported.     

Drosophila suzukii wing spot size is robust to developmental temperature

Phenotypic plasticity is an important mechanism allowing adaptation to new environments and as such it has been suggested to facilitate biological invasions. Under this assumption, invasive populations are predicted to exhibit stronger plastic responses than native populations. Drosophila suzukii is an invasive species whose males harbor a spot on the wing tip. In this study, by manipulating developmental temperature, we compare the phenotypic plasticity of wing spot size of two invasive populations with that of a native population. We then compare the results with data obtained from wild‐caught flies from different natural populations. While both wing size and spot size are plastic to temperature, no difference in plasticity was detected between native and invasive populations, rejecting the hypothesis of a role of the wing‐spot plasticity in the invasion success. In contrast, we observed a remarkable stability in the spot‐to‐wing ratio across temperatures, as well as among geographic populations. This stability suggests either that the spot relative size is under stabilizing selection, or that its variation might be constrained by a tight developmental correlation between spot size and wing size. Our data show that this correlation was lost at high temperature, leading to an increased variation in the relative spot size, particularly marked in the two invasive populations. This suggests: (a) that D. suzukii's development is impaired by hot temperatures, in agreement with the cold‐adapted status of this species; (b) that the spot size can be decoupled from wing size, rejecting the hypothesis of an absolute constraint and suggesting that the wing color pattern might be under stabilizing (sexual) selection; and (c) that such sexual selection might be relaxed in the invasive populations. Finally, a subtle but consistent directional asymmetry in spot size was detected in favor of the right side in all populations and temperatures, possibly indicative of a lateralized sexual behavior.     

Seasonal polyphenism in wing coloration affects species recognition in rubyspot damselflies (Hetaerina spp.)

Understanding how phenotypic plasticity evolves and in turn affects the course of evolution is a major challenge in modern biology. By definition, biological species are reproductively isolated, but many animals fail to distinguish between conspecifics and closely related heterospecifics. In some cases, phenotypic plasticity may interfere with species recognition. Here, we document a seasonal polyphenism in the degree of dark wing pigmentation in smoky rubyspot damselflies (Hetaerina titia) – a shift so pronounced that it led early researchers to classify different forms of H. titia as separate species. We further show how the seasonal colour shift impacts species recognition with the sympatric congener Hetaerina occisa. Interspecific aggression (territorial fights) and reproductive interference (mating attempts) are much more frequent early in the year, when H. titia more closely resembles H. occisa, compared to later in the year when the dark phase of H. titia predominates. Using wing colour manipulations of tethered damselflies, we show that the seasonal changes in interspecific interactions are caused not only by the seasonal colour shift but also by shifts in discriminatory behaviour in both species. We also experimentally tested and rejected the hypothesis that learning underlies the behavioural shifts in H. occisa. An alternative hypothesis, which remains to be tested, is that the seasonal polyphenism in H. titia wing coloration has resulted in the evolution of a corresponding seasonal polyphenism in species recognition in H. occisa. This study illustrates one of the many possible ways that plasticity in species recognition cues may influence the evolution of interspecific interactions.     

Do reproductive activities compromise immunological competence as measured by phenoloxidase activity? Field and experimental manipulation in females of two damselfly species

Recent studies of female insects indicate that reproductive activities, such as mating and oviposition, can impair immune ability. Using the two tropical damselfly species Argia anceps Garrison and Hetaerina americana (Fabricius), egg production and phenoloxidase (PO) activity, a key enzyme in insect immunity, are measured in mating, ovipositing and perching females in December and March. Perching females of both species have fewer eggs compared with mating and ovipositing females, which suggests that perching females are not engaged in reproduction. There is seasonal variation in egg number for the three categories in H. americana but not in A. anceps, which can be interpreted in terms of adaptive changes in egg production depending on female–male interactions in the former species but not in the latter species. There is no difference in PO activity among mating, ovipositing or perching females within either species, although measurements in December and March indicate distinct seasonal changes. Juvenile Hormone (JH) is known to reduce the effectiveness of the immune system by favouring the use of resources for reproduction. A possible role for JH is examined in H. americana, using the JH analogue methoprene to manipulate hormone activity, revealing that PO activity is reduced in methoprene‐treated H. americana females. Thus, although the results of the present study are indicative of possible hormone‐driven changes in PO, there is not necessarily a down‐regulation of immune function (as determined by PO activity) during mating or oviposition. The results complement some recent studies countering the idea that reproductive activities reduce the immune ability in insects.     

Seasonal variability of wing length and eyespots in populations of <Emphasis Type="Italic">Erebia ligea</Emphasis> (L.) (Lepidoptera,Satyridae) in the Middle Urals

The flight period of Erebia ligea (Linnaeus, 1758) in the Sysert District of Sverdlovsk Province lasts from early July until early August only in the odd-numbered years. The species is characterized by protandry, its males emerging 5–7 days before females. Erebia ligea manifests sexual and seasonal variation of the wing length and diameter of the eyespots in the Middle Urals. According to the literature and newly obtained data, the relative male/female wing length varies within the species range. The vectors of the seasonal variation of the body size are opposite in the males and females: in early summer the largest males and the smallest females occur, then mediumsized individuals appear, while the smallest males and the largest females fly at the end of the warm period. Although the males of Erebia ligea are larger than females, the latter have larger eyespots on the wings. Seasonal variation of the diameters of the forewing eyespots is largely similar in the two sexes: individuals with larger spots appear earlier in the season than those with small spots. The eyespots treated as phenes in this study develop on the wings only if the remaining spots of the wing pattern attain a threshold size. A threshold nature was shown for P4 spot in M ₃-Cu ₁ cell of the fore wing and G2 spot in cell M ₁–M ₂ of the hind wing. Individuals with and without phene G2 have opposite vectors of seasonal variation of the sizes of the rest spots of the hind wing pattern. It is apparently the threshold character of variation of these elements of the wing pattern that defines the variation of the proportion of phenetic combinations of the eyespots in the individuals emerging in different periods of the season. Describing phenetic variation at the population level, one should remember that the percentage of the “morphs” or phenetic combinations may vary during a season, especially in the protandric species.     

Sympatric shift in a male sexual ornament in the damselfly Calopteryx splendens

Character displacement is a process by which interactions between two species that exhibit similar traits, results in geographical patterns of trait divergence in one or both species. These traits evolve to reduce costs of interspecific interactions in sympatry and thus differ from their condition in allopatry. In male damselflies Calopteryx splendens, large wing spots are sexually selected. However, in sympatric populations with Calopteryx virgo, wing spot size decreases as C. virgo abundance increases. The stability of this pattern is unclear, because previous studies have focused on sympatric populations with potentially fluctuating relative abundances. We studied the wing spot sizes of C. splendens in both sympatric and allopatric populations. Our data show that male C. splendens’ wing spots are larger in allopatry than in sympatry with C. virgo. We suggest that both interspecific aggression and avoidance of interspecific reproductive interactions may result in this pattern, although their relative importance remains unclear.     

Growth temperature and phenotypic plasticity in two Drosophila sibling species: probable adaptive changes in flight capacities

In the sibling species Drosophila melanogaster and D. simulans, growth and development at constant temperatures, from 12 to 30 °C, resulted in extensive variations of adult size and flight parameters with significant differences between species. Changes in body weight, thorax length and wing length were nonlinear, with maximum values of each trait at lower temperatures for D. simulans than for its sibling species. By contrast, the wing/thorax ratio and the wing loading varied monotonically with growth temperature. These traits were negatively correlated, the wing/thorax ratio decreasing with growth temperature while the wing loading increased. Wing/thorax ratio, which is easier to measure, thus appears as a convenient predictor of wing loading. During tethered flight at the same ambient temperature, the wingbeat frequency changed linearly as a function of the wing moment of inertia. More interestingly, the beat rate was strongly correlated with the increase of wing loading at growth temperature above 13 °C. The likely adaptive significance of these morphometrical changes for flight efficiency is discussed.     

Wing Colour Properties do not Reflect Male Condition in the American Rubyspot (Hetaerina americana)

Adult males of the American rubyspot ( Hetaerina americana ) dispute riverine territories where females arrive to mate. On the wing basis, these males bear a red pigmentation spot whose area correlates with territorial disputes and mating rate: males with larger spots are more successful. This is explained by the fact that spot size correlates with fat muscular reserves which fuel flight during territorial intrusions. To further our understanding of sexual selection acting on the spot, here we have examined possible differences in three spot colour properties (red chroma, hue and brightness) in three distinct adult male ages [young, middle-aged (when males are more likely to defend a territory) and old], social status (territorial and non-territorial in middle-aged males), and under two potentially, energetically and costly situations: when faced with an immune challenge [comparing a nylon-implanted male group vs. a non-implanted male group in two ages, teneral (previous to colour formation) and middle-aged] and low diet levels (comparing a male set of middle-aged animals that received food ad libitum vs. a male set that received no food). Our results indicate no change in colour properties across any of these comparisons. Taken together, these and previous results suggest that only spot size but not the spot characteristics we measured here, is sexually selected in males of this species at least in terms of pre-copulatory male–male competition. That some of these colour properties have been related to male condition in other calopterygid damselflies cannot be generalized to the American rubyspot.     

The Role of Wing Coloration in Sex Recognition and Competitor Recognition in Rubyspot Damselflies (Hetaerina spp.)

The decision rules that animals use for distinguishing between conspecifics of different age and sex classes are relevant for understanding how closely related species interact in sympatry. In rubyspot damselflies (Hetaerina spp.), the red wing coloration of mature males is hypothesized to be a key trait for sex recognition and competitor recognition within species and the proximate trigger for interspecific male–male aggression. We tested this hypothesis by manipulating the wing coloration of tethered conspecific intruders and measuring the responses of territory holders of three species in the field. As predicted, covering the red spots of mature males with black ink nearly eliminated territorial responses, and in some cases, territorial holders clasped the blackened males as if they were females. Adding red spots to female wings triggered territorial responses and nearly eliminated sexual responses. Immature males with artificial red spots were attacked at the same rate as mature male intruders, and much more frequently than were immature male controls. The results varied somewhat by species. In H. titia, the only species of Hetaerina with substantial black wing pigmentation, the effects of blackening the red spots of intruders varied both geographically and seasonally. But even when blackening the red spots of male intruders did not reduce the aggressive response of H. titia territory holders, adding artificial red spots to female wings elicited aggressive responses and nearly eliminated sexual responses. The results of this study further strengthen the evidence that interspecific aggression in Hetaerina results from overlap in territorial signals and that the derived black wing pigmentation of H. titia reduces interspecific aggression.     

Butterfly Wings Are Three-Dimensional: Pupal Cuticle Focal Spots and Their Associated Structures in Junonia Butterflies

Butterfly wing color patterns often contain eyespots, which are developmentally determined at the late larval and early pupal stages by organizing activities of focal cells that can later form eyespot foci. In the pupal stage, the focal position of a future eyespot is often marked by a focal spot, one of the pupal cuticle spots, on the pupal surface. Here, we examined the possible relationships of the pupal focal spots with the underneath pupal wing tissues and with the adult wing eyespots using Junonia butterflies. Large pupal focal spots were found in two species with large adult eyespots, J. orithya and J. almana, whereas only small pupal focal spots were found in a species with small adult eyespots, J. hedonia. The size of five pupal focal spots on a single wing was correlated with the size of the corresponding adult eyespots in J. orithya. A pupal focal spot was a three-dimensional bulge of cuticle surface, and the underside of the major pupal focal spot exhibited a hollowed cuticle in a pupal case. Cross sections of a pupal wing revealed that the cuticle layer shows a curvature at a focal spot, and a positional correlation was observed between the cuticle layer thickness and its corresponding cell layer thickness. Adult major eyespots of J. orithya and J. almana exhibited surface elevations and depressions that approximately correspond to the coloration within an eyespot. Our results suggest that a pupal focal spot is produced by the organizing activity of focal cells underneath the focal spot. Probably because the focal cell layer immediately underneath a focal spot is thicker than that of its surrounding areas, eyespots of adult butterfly wings are three-dimensionally constructed. The color-height relationship in adult eyespots might have an implication in the developmental signaling for determining the eyespot color patterns.