Rubyspot Damselflies Research Articles

Species distribution models predict genetic isolation of Hetaerina vulnerata Hagen in Selys, 1853 (Odonata, Calopterygidae).

Understanding how past and current environmental conditions shape the demographic and genetic distributions of organisms facilitates our predictions of how future environmental patterns may affect populations. The Canyon Rubyspot damselfly (Odonata: Zygoptera: Hetaerina vulnerata) is an insect with a range distribution from Colombia to the arid southwestern United States, where it inhabits shaded mountain streams in the arid southwestern United States. Past spatial fragmentation of habitat and limited dispersal capacity of H. vulnerata may cause population isolation and genetic differentiation, and projected climate change may exacerbate isolation by further restricting the species' distribution. We constructed species distribution models (SDMs) based on occurrences of H. vulnerata and environmental variables characterizing the species' niche. We inferred seven current potential population clusters isolated by unsuitable habitat. Paleoclimate models indicated habitat contiguity in past conditions; projected models indicated some habitat fragmentation in future scenarios. Seventy-eight H. vulnerata individuals from six of the current clusters were sequenced via ddRADseq and processed with Stacks. Principal components and phylogeographic analyses resolved three subpopulations; Structure resolved four subpopulations. F ST values were low (<0.05) for nearby populations and >0.15 for populations separated by expanses of unsuitable habitat. Isolation by distance was an existing but weak factor in determining genomic structure; isolation by environment and the intervening landscape explained a significant proportion of genetic distance. Hetaerina vulnerata populations were shown to be isolated by a lack of tree canopy coverage, an important habitat predictor for oviposition and territoriality. Thus, H. vulnerata populations are likely separated and are genetically isolated. Integrating SDMs with landscape genetics allowed us to identify populations separated by distance and unsuitable habitat, explaining population genetic patterns and probable fates for populations under future climate scenarios.

Niche differentiation, reproductive interference, and range expansion.

Understanding species distributions and predicting future range shifts requires considering all relevant abiotic factors and biotic interactions. Resource competition has received the most attention, but reproductive interference is another widespread biotic interaction that could influence species ranges. Rubyspot damselflies (Hetaerina spp.) exhibit a biogeographic pattern consistent with the hypothesis that reproductive interference has limited range expansion. Here, we use ecological niche models to evaluate whether this pattern could have instead been caused by niche differentiation. We found evidence for climatic niche differentiation, but the species that encounters the least reproductive interference has one of the narrowest and most peripheral niches. These findings strengthen the case that reproductive interference has limited range expansion and also provide a counterexample to the idea that release from negative species interactions triggers niche expansion. We propose that release from reproductive interference enables species to expand in range while specializing on the habitats most suitable for breeding.

A chromosome-level genome assembly for the smoky rubyspot damselfly (Hetaerina titia).

Smoky rubyspot damselflies (Hetaerina titia Drury, 1773) are one of the most commonly encountered odonates along streams and rivers on both slopes of Central America and the Atlantic drainages in the United States and southern Canada. Owing to their highly variable wing pigmentation, they have become a model system for studying sexual selection and interspecific behavioral interference. Here, we sequence and assemble the genome of a female smoky rubyspot. Of the primary assembly (i.e. the principle pseudohaplotype), 98.8% is made up of 12 chromosomal pseudomolecules (2N = 22A + X). There are 75 scaffolds in total, an N50 of 120 Mb, a contig-N50 of 0.64 Mb, and a high arthropod BUSCO score [C: 97.6% (S: 97.3%, D: 0.3%), F: 0.8%, M: 1.6%]. We then compare our assembly to that of the blue-tailed damselfly genome (Ischnura elegans), the most complete damselfly assembly to date, and a recently published assembly for an American rubyspot damselfly (Hetaerina americana). Collectively, these resources make Hetaerina a genome-enabled genus for further studies of the ecological and evolutionary forces shaping biological diversity.

Is body condition of Mexican rubyspot (Odonata:Zygoptera) associated with urbanization?

Urbanized areas can impose selective pressures on insects which can be identified at the individual level based on animal physiological condition. Physiological condition can be measured from variables such as body size, body mass or energetic budget of individuals. We examined whether body mass, wing spot size and energy reserves (proteins, lipids and carbohydrates) were reduced as urbanization increases, using adults of two damselflies, Hetaerina americana and H. vulnerata, in Central Mexico. We used a Habitat Integrity Index to assess the degree of urbanization in our sampled sites, considering biophysical attributes and the impact of anthropogenic activities. We did not find relation of above individual variables with urbanization degree. These results support possible resilience of rubyspot damselflies in the face of radical changes such as urbanization. Our finding echoes other results in damselflies research.Implications for insect conservationOur results highlight the resilience of these damselflies species in the face of urban disturbances. Thus design cities considering not only requirements of humans is essential to promote the presence and conservation of these and other species of insects in cities.Graphical abstract

Mechanisms of reduced interspecific interference between territorial species

AbstractInterspecific territoriality has complex ecological and evolutionary consequences. Species that interact aggressively often exhibit spatial or temporal shifts in activity that reduce the frequency of costly encounters. We analyzed data collected over a 13-year period on 50 populations of rubyspot damselflies (Hetaerina spp.) to examine how rates of interspecific fighting covary with fine-scale habitat partitioning and to test for agonistic character displacement in microhabitat preferences. In most sympatric species, interspecific fights occur less frequently than expected based on the species’ relative densities. Incorporating measurements of spatial segregation and species discrimination into the calculation of expected frequencies accounted for most of the reduction in interspecific fighting (subtle differences in microhabitat preferences could account for the rest). In 23 of 25 sympatric population pairs, we found multivariate differences between species in territory microhabitat (perch height, stream width, current speed, and canopy cover). As predicted by the agonistic character displacement hypothesis, sympatric species that respond more aggressively to each other in direct encounters differ more in microhabitat use and have higher levels of spatial segregation. Previous work established that species with the lowest levels of interspecific fighting have diverged in territory signals and competitor recognition through agonistic character displacement. In the other species pairs, interspecific aggression appears to be maintained as an adaptive response to reproductive interference, but interspecific fighting is still costly. We now have robust evidence that evolved shifts in microhabitat preferences also reduce the frequency of interspecific fighting.

Complex evolutionary history of the American Rubyspot damselfly, Hetaerina americana (Odonata): Evidence of cryptic speciation

Analyzing the magnitude and distribution of genetic variation within and among populations allows for hypothesis testing about historical demographic size changes, secondary contacts, refugia, and speciation patterns. Species distribution and genetic structure are greatly influenced by the complex life cycle and behavior of odonates. Hetaerina americana has been widely used as a model system in behavioral studies, but its population genetic structure has not been analyzed, except for a single study that included only three populations but identified the presence of markedly differentiated genetic groups, suggesting the existence of cryptic species. Here, we tested this hypothesis by assessing throughout the distribution range of H. americana the patterns of genetic and morphological variation in the male caudal appendages, due to the great importance of these structures in mate recognition. As molecular markers we used sequences of the mitochondrial cytochrome oxidase I (COI) gene and the nuclear internal transcribed spacer (ITS) region, as well as six nuclear microsatellites. We found very high population genetic differentiation (ΦST > 0.51) in the three sets of markers but with strong mitonuclear discordance. A neutrality test suggested that the mitochondrial genome might be under purifying selection in association to climatic variables (temperature seasonality). The assignment of individuals to nuclear genetic groups showed little admixture and complete congruence with morphological differentiation in the male caudal appendages. Hence, the results suggest that H. americana represents at least two different cryptic species which are isolated reproductively.

A General Explanation for the Persistence of Reproductive Interference.

Reproductive interference is widespread, despite the theoretical expectation that it should be eliminated by reproductive character displacement (RCD). A possible explanation is that females of sympatric species are too similar phenotypically for males to distinguish between them, resulting in a type of evolutionary dilemma or catch-22 in which reproductive interference persists because male mate recognition (MR) cannot evolve until female phenotypes diverge further, and vice versa. Here we illustrate and test this hypothesis with data on rubyspot damselflies (Hetaerina spp.). First, reproductive isolation owing to male MR breaks down with increasing interspecific similarity in female phenotypes. Second, comparing allopatric and sympatric populations yielded no evidence for RCD, suggesting that parallel divergence in female coloration and male MR in allopatry determines the level of reproductive isolation on secondary contact. Whenever reproductive isolation depends on male MR and females of sympatric species are phenotypically similar, the evolutionary catch-22 hypothesis offers an explanation for the persistence of reproductive interference.

Assortative Mating by Size in the American Rubyspot Damselfly (Hetaerina americana)

Assortative mating refers to the non-random nature of mating patterns between certain males and females. Thus, males and females may associate negative- or positively, based on different traits. Amongst these associations, assortative mating by size is one of the most common patterns found in natural populations of animals. Two main hypotheses have been proposed to account for the occurrence of assortative mating by size. First, it may be the result of mechanical, temporal, or physiological constraints. Second, it may occur in response to direct or indirect selection on mating preferences. Here we investigate whether the American rubyspot damselfly exhibits true assortative mating by size. Males of this species exhibit high levels of male-male competition, as they compete over territories, to which females are attracted for copulation. There is a documented large male body size advantage: the largest males are better able to hold their territories and thus secure more copulations. Our major results show that i) mated males are more likely to be larger than unmated males, whereas mated and unmated females tend to have similar body sizes; ii) H. americana exhibits true assortative mating by size; as such, this pattern is not driven by seasonal changes in the body sizes of males and females. We suggest that this mating pattern occurs in this species given the advantages of large male size, and the advantages of large female body size (i.e. higher fecundity). We believe that males may be able to evaluate a female’s reproductive value and exert mate choice.

Body temperature regulation is associated with climatic and geographical variables but not wing pigmentation in two rubyspot damselflies (Odonata: Calopterygidae)

AbstractIt has been proposed that wing pigmented spots function in temperature control in male calopterygids. Using two rubyspot species Hetaerina americana Fabricius and Hetaerina vulnerata Hagen in Selys, the present study investigated whether (i) wing spot size and colour‐modified aspect can predict temperature gain after a cooling event; (ii) wing spot size is related to the temperature needed to fly and how long it takes to initiate flight; and (iii) wing spot size is related to seasonality and altitude. The results obtained do not support any of these relationships. The results also indicate that H. vulnerata can achieve flight at 8 °C less than H. americana. The present study further investigates whether the species differ in their latitudinal and geographical distribution, and respond differently to bioclimatic variables. The results obtained provide support for this particular hypothesis, showing that H. vulnerata inhabits higher altitudes, and is able to tolerate colder environments compared with H. americana. Wing spots in the two Hetaerina species do not help in thermoregulation, although both species show different temperature control abilities. This difference in thermoregulatory ability may enable the species to colonize different environments and reduce interspecific competition.

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.

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

AbstractThe 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.

Interspecific aggression, not interspecific mating, drives character displacement in the wing coloration of male rubyspot damselflies (Hetaerina).

Traits that mediate intraspecific social interactions may overlap in closely related sympatric species, resulting in costly between-species interactions. Such interactions have principally interested investigators studying the evolution of reproductive isolation via reproductive character displacement (RCD) or reinforcement, yet in addition to reproductive interference, interspecific trait overlap can lead to costly between-species aggression. Previous research on rubyspot damselflies (Hetaerina spp.) demonstrated that sympatric shifts in male wing colour patterns and competitor recognition reduce interspecific aggression, supporting the hypothesis that agonistic character displacement (ACD) drove trait shifts. However, a recent theoretical model shows that RCD overshadows ACD if the same male trait is used for both female mate recognition and male competitor recognition. To determine whether female mate recognition is based on male wing coloration in Hetaerina, we conducted a phenotype manipulation experiment. Compared to control males, male H. americana with wings manipulated to resemble a sympatric congener (H. titia) suffered no reduction in mating success. Thus, female mate recognition is not based on species differences in male wing coloration. Experimental males did, however, experience higher interspecific fighting rates and reduced survival compared to controls. These results greatly strengthen the case for ACD and highlight the mechanistic distinction between ACD and RCD.

Quantifying the refractive index dispersion of a pigmented biological tissue using Jamin–Lebedeff interference microscopy

Scientists have used Jamin–Lebedeff interference microscopy to measure the refractive index of strongly pigmented biological tissue. Doekele Stavenga and co-workers from the University of Groningen in the Netherlands used the technique to analyze the refractive index, and its variation with wavelength, of the red wings of the American Rubyspot Damselfly. Until now, this microscopy scheme has been applied only to transparent, unpigmented tissue. The researchers report that the high density of pigment in the damselfly wing results in a significant anomalous dispersion of its refractive index, with the index increasing for longer wavelengths. This is believed to enhance the reflectivity in the red spectral region and thus improve the colour contrast of the wing decorations. Similar contrast shown effects are thought to be present in the eyes of some crustaceans.

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

The effects of food shortage during larval development on adult body size, body mass, physiology and developmental time in a tropical damselfly

Few studies have looked jointly at the effects of larval stressors on life history and physiology across metamorphosis, especially in tropical insects. Here we investigated how the variation of food availability during the larval stage of the tropical and territorial American rubyspot damselfly (Hetaerina americana) affects adult body size and body mass, and two physiological indicators of condition – phenoloxidase activity (an indicator of immune ability) and protein concentration. We also investigated whether larval developmental time is prolonged when food is scarce, an expected situation for tropical species whose larval time is less constrained, compared to temperate species. Second instar larvae were collected from their natural environments and reared in one of two diet regimes: (i) “rich” provided with five Artemiasalina prey every day, and (ii) “poor” provided with two A. salina prey every day. In order to compare how distinct our treatments were from natural conditions, a second set of last-instar larvae were also collected and allowed to emerge. Only body size and phenoloxidase increased in the rich regime, possibly to prioritize investment on sexually selected traits (which increase mating opportunities), and immune ability, given pathogen pressure. The sexes did not differ in body size in relation to food regimes but they did differ in body mass and protein concentration; this can be explained on the basis of the energetically demanding territorial activities by males (for the case of body mass), and female allocation to egg production (for the case of protein). Finally, animals delayed larval development when food was scarce, which is coherent for tropical environments. These findings provide key insights in the role of food availability in a tropical species.

Nubenocephalus nickoli n. sp. and Nubenocephalus xunantunichensis n. sp. (Apicomplexa: Eugregarinida: Actinocephalidae) Parasitizing Damselflies (Odonata: Zygoptera) in Belize, Central America

Two new species of Nubenocephalus (Apicomplexa: Eugregarinida: Actinocephalidae) are described from adult damselflies sampled in Cayo District, Belize, Central America. Nubenocephalus nickoli n. sp. is described from the American Rubyspot damselfly, Hetaerina americana, and the Smoky Rubyspot damselfly, Hetaerina titia (Odonata: Zygoptera: Hetaerinidae), and Nubenocephalus xunantunichensis n. sp. is described from the Comanche Dancer, Argia chelata (Odonata: Zygoptera: Coenagrionidae). New, complete morphological data sets are reported for populations of Nubenocephalus nebraskensis and Nubenocephalus secundus, and a complete, synoptic reevaluation of diagnostic morphological characters used to discriminate species within Nubenocephalus is presented for all known species of the genus in the New World. Nubenocephalus nebraskensis is reported from Argia apicalis for the first time.

Male mating tactics in the American rubyspot damselfly: territoriality, nonterritoriality and switching behaviour

Odonates exhibit a wide range of territorial and nonterritorial mating tactics and are ideal for investigating alternative reproductive behaviours. We studied male mating tactics in the American rubyspot damselfly, Hetaerina americana , a species that exhibits red wing spots that have been suggested to have evolved as a consequence of male–male contests. In this species mating success is enhanced by the ability of males to defend territories along streams and rivers, which depends on the amount of thoracic fat reserves available. Previous studies on this species have distinguished between territorial and nonterritorial males, in which the former obtain significantly more matings than the latter. In our study, however, we found a third reproductive tactic: switching. Switcher males exhibit both territorial and nonterritorial tactics and a mating success similar to that of territorial and nonterritorial males, although this result may be confounded by the small sample size used for this analysis. We suggest that the different mating tactics may be condition determined: territorial males contained the highest fat reserves, nonterritorial males had the least fat content and switchers had intermediate fat loads. We also show that there were no age differences between males using these tactics. Our results suggest that territorial behaviour is extremely plastic in this species. Finally, we discuss the implications of our study and directions for future work on territorial and nonterritorial reproductive tactics in odonates.

The expression of a sexually selected trait correlates with different immune defense components and survival in males of the American rubyspot

Recent studies have suggested that courtship trait expression indicates immune strength. However, most studies have measured only one immune parameter, have not assessed individual differences in immune ability according to time and have not controlled for ecological differences among individuals after an immune challenge. In this work, we tested this hypothesis and controlled for these factors using males of the American rubyspot damselfly which bear a wing red spot whose size is evolutionarily maintained via male–male territorial competition. Our general hypothesis was that territorial, large-spotted males, had a better immune ability compared to nonterritorial, small-spotted males. We expected that the following variables were greater in territorial males compared to nonterritorial males: spot size, phenoloxidase (PO) and hydrolytic enzymatic (HE) activity in males challenged and nonchallenged with a nylon implant, PO and HE activity rate; PO activity after a Serratia marcescens challenge, and survival after a nylon challenge controlling for activity and feeding differences. We found that territorial males showed larger spot areas, greater PO and HE activity (independently of whether they were challenged or not), a higher rate of PO and HE activity (but only expressed at 8 h), greater PO production after the bacterial challenge, and a higher survival after the challenge. These results corroborate that males with more pronounced sexual traits have a superior immune function.

Mechanisms for the formation and maintenance of traditional night roost aggregations in a territorial damselfly

Communal roosting has been studied extensively in birds, but the mechanisms and functions of this taxonomically widespread behaviour pattern remain poorly understood. We studied the roosting behaviour of rubyspot damselflies,Hetaerina americana , in relation to sex and territorial status, and conducted field experiments to test for specific mechanisms of roost formation and maintenance. Both sexes tended to return close to their previous night's roost, but only males were significantly more roost site faithful than chance expectations based on individual day ranges. Males were more roost site faithful when they held mating territories. After acquiring a territory, males usually began roosting closer to the territory after a delay of a few days. Roosts were not located at sites that reduced the daily commuting distance between hunting areas and territories; males generally hunted closer to their territories than to their roosts. In field experiments, sites ‘seeded’ with synthetic models of male rubyspots attracted more recruits than vacant control sites and control sites seeded with nonrubyspot (clear-winged) damselfly models. Sites seeded repeatedly with rubyspot models often remained popular for roosting after the models were removed, suggesting that the models established new traditional roosts. These results indicate that conspecific attraction and individual spatial memory together may be sufficient to explain, at a proximate level, the traditional night roost aggregations of this species. We discuss these results in relation to functional hypotheses for roost site choice and fidelity.

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.