Hetaerina Americana Research Articles

Changes in effective population size of Odonata in response to climate change revealed through genomics

The advent of third generation sequencing technologies has led to a boom of high-quality, chromosome level genome assemblies of Odonata, but to date, these have not been widely used to estimate the demographic history of the sequenced species through time. Yet, an understanding of how lineages have responded to past changes in the climate is useful in predicting their response to current and future changes in the climate. Here, we utilized the pairwise sequential markovian coalescent (PSMC) to estimate the demographic histories of Sympetrum striolatum, Ischnura elegans, and Hetaerina americana, three Odonata for which chromosome-length genome assemblies are available. Ischnura elegans showed a sharp decline in effective population size around the onset of the Pleistocene ice ages, while both S. striolatum and H. americana showed more recent declines. All three species have had relatively stable population sizes over the last one hundred thousand years. Although it is important to remain cautious when determining the conservation status of species, the coalescent models did not show any reason for major concern in any of the three species tested. The model for I. elegans confirmed prior research suggesting that population sizes of I. elegans will increase as temperatures rise.

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.

Genomic differentiation and niche divergence in the Hetaerina americana (Odonata) cryptic species complex.

The evolution of reproductive barriers, that is, the speciation process, implies the limitation of gene flow between populations. Different patterns of genomic differentiation throughout the speciation continuum may provide insights into the causal evolutionary forces of species divergence. In this study, we analysed a cryptic species complex of the genus Hetaerina (Odonata). This complex includes H. americana and H. calverti; however, in H. americana two highly differentiated genetic groups have been previously detected, which, we hypothesize, may correspond to different species with low morphological variation. We obtained single nucleotide polymorphism (SNP) data for 90 individuals belonging to the different taxa in the complex and carried out differentiation tests to identify genetic isolation. The results from STRUCTURE and discriminant analysis of principal components (DAPC), based on almost 5000 SNPs, confirmed the presence of three highly differentiated taxa. Also, we found FST values above 0.5 in pairwise comparisons, which indicates a considerable degree of genetic isolation among the suggested species. We also found low climatic niche overlap among all taxa, suggesting that each group occurs at specific conditions of temperature, precipitation and elevation. We propose that H. americana comprises two cryptic species, which may be reproductively isolated by ecological barriers related to niche divergence, since the morphological variation is minimal and, therefore, mechanical barriers are probably less effective compared to other related species such as H. calverti.

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

Reference genome for the American rubyspot damselfly, Hetaerina americana.

Damselflies and dragonflies (Order: Odonata) play important roles in both aquatic and terrestrial food webs and can serve as sentinels of ecosystem health and predictors of population trends in other taxa. The habitat requirements and limited dispersal of lotic damselflies make them especially sensitive to habitat loss and fragmentation. As such, landscape genomic studies of these taxa can help focus conservation efforts on watersheds with high levels of genetic diversity, local adaptation, and even cryptic endemism. Here, as part of the California Conservation Genomics Project (CCGP), we report the first reference genome for the American rubyspot damselfly, Hetaerina americana, a species associated with springs, streams and rivers throughout California. Following the CCGP assembly pipeline, we produced two de novo genome assemblies. The primary assembly includes 1,630,044,487 base pairs, with a contig N50 of 5.4 Mb, a scaffold N50 of 86.2 Mb, and a BUSCO completeness score of 97.6%. This is the seventh Odonata genome to be made publicly available and the first for the subfamily Hetaerininae. This reference genome fills an important phylogenetic gap in our understanding of Odonata genome evolution, and provides a genomic resource for a host of interesting ecological, evolutionary, and conservation questions for which the rubyspot damselfly genus Hetaerina is an important model system.

Morphological variation and reproductive isolation in the Hetaerina americana species complex

Incomplete premating barriers in closely related species may result in reproductive interference. This process has different fitness consequences and can lead to three scenarios: niche segregation, sexual exclusion, or reproductive character displacement. In morphologically cryptic species, isolation barriers can be difficult to recognize. Here, we analyzed the morphological, behavioral, and genetic differences between two sympatric cryptic species of the genus Hetaerina to determine the characters that contribute the most to reproductive isolation and the effect of the high rates of behavior interference between the species. We found complete genetic isolation and significant differences in the morphometry of caudal appendages and wing shape, as well as body size variation between species. In contrast, we did not find clear differences in the coloration of the wing spot and observed high rates of interspecific aggression. Our results suggest that divergence in the shape of the caudal appendages is the principal pre-mating barrier that prevents interspecific mating. Moreover, a scenario of character displacement on body size was found. Nevertheless, size could play an important role in both inter- and intrasexual interactions and, therefore, we cannot differentiate if it has resulted from reproductive or aggressive interference.

What doesn't kill you makes you stronger: Detoxification ability as a mechanism of honesty in a sexually selected signal

Abstract Sexual selection leads to the expression and maintenance of colourful signals. The metabolic pathways to produce such signals often involve toxic byproducts that can reduce survival. However, rather than discarding these otherwise harmful byproducts, animals may use them by integrating them into sexually selected traits. We tested this using the damselfly Hetaerina americana, where males bear a red wing spot (RWS) that has evolved by intrasexual competition. By using confocal microscopy and several biochemical techniques, we determined that the RWS are generated by ommochrome pigments derived from tryptophan metabolism. Second, we injected a group of males with the toxic precursor of these ommochromes, 3‐hydroxy‐kynurenine (3‐Hk), confirming the toxicity of this compound in adult males. Finally, we showed that adult males injected with a median lethal concentration of 3‐Hk had more ommochromes in their RWS than controls but similar survival, suggesting that the deposition of ommochrome pigment in the wing counteracts the 3‐HK toxicity. Thus, we report that sexually selected pigmented signals may involve the co‐option of excreted compounds that could otherwise have lethal effects, a hypothesis we call ‘detoxifying ability signalling’. Our results provide new insights about the evolution of sexual signals, elucidating a mechanism for the evolution of honest indicators of quality that could have arisen due to natural selection.

Hetaerina calverti (Odonata: Zygoptera: Calopterygidae) sp. nov., a new cryptic species of the American Rubyspot complex.

Hetaerina americana Fabricius, 1798 has a long and irresolute taxonomic history. Several synonyms have been suggested (H. californica Hagen in Selys-Longchamps, 1859, H. basalis Hagen in Selys-Longchamps, 1859, H. texana Walsh, 1863, H. scelerata Walsh, 1863, etc.), related to the variation in the size of the wing spots as well as to the morphology of the male cerci. However, Calvert (1901) suggested that H. americana represents one variable species. Nevertheless, Vega-Sánchez et al. (2019) through a genetic and morphological analysis presented evidence to propose that H. americana represents a species complex. In the present work, we describe a new species that belongs to this complex: H. calverti sp. nov. The morphological characteristics by which males and females of H. calverti differ from H. americana are highlighted. The most important character for the differentiation of males is the shape of the cerci and the size of the individuals (when the two species are in sympatry). In females, the main differences are in the shape of the intersternites and the medio-dorsal carina of the last segment of the abdomen. Some generalities about the biology of the species are presented, including geographical distribution patterns and genetic divergence data. [urn:lsid:zoobank.org:act:F5C329AE-7A00-4979-8A0D-A13D869E54B1].

Measuring the refractive index dispersion of (un)pigmented biological tissues by Jamin-Lebedeff interference microscopy

Jamin-Lebedeff interference microscopy is a powerful technique for measuring the refractive index of microscopically-sized solid objects. This method was classically used for transparent objects immersed in various refractive-index matching media by applying light of a certain predesigned wavelength. In previous studies, we demonstrated that the Jamin-Lebedeff microscopy approach can also be utilized to determine the refractive index of pigmented media for a wide range of wavelengths across the visible spectrum. The theoretical basis of the extended method was however only precise for a single wavelength, dependent on the characteristics of the microscope setup. Using Jones calculus, we here present a complete theory of Jamin-Lebedeff interference microscopy that incorporates the wavelength-dependent correction factors of the half- and quarter-wave plates. We show that the method can indeed be used universally in that it allows the assessment of the refractive index dispersion of both unpigmented and pigmented microscopic media. We illustrate this on the case of the red-pigmented wing of the damselfly Hetaerina americana and find that very similar refractive indices are obtained whether or not the wave-plate correction factors are accounted for.

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.

Damselfly (Odonata: Calopterygidae) Population Decline in an Urbanizing Watershed.

Reduction of terrestrial vegetation and degradation of water quality are among the factors driving insect population decline in growing cities. In this study, we investigated the extent of habitat deterioration, behavioral and physiological responses, and fitness of a damselfly [Hetaerina americana (F.)] population in a semitropical region in central Mexico. The study population was located in a riverine area that crosses a small urban area (Tehuixtla city). We related two habitat variables (tree/shrub covered area and numbers of wastewater outlets) to presumable damselfly responses (larval and adult abundance, duration of adults exposed directly to sunlight, lipid content and muscle mass, and egg survival) over the years 2002 and 2016. We detected a reduction in terrestrial vegetation cover, an increase in wastewater outlets, and a decrease in larval and adult abundance. Adults were more exposed to sunlight in 2016 than in 2002 and showed a reduced lipid content and muscle mass in 2016. Egg survival also decreased. Although correlative, these results suggest impairment of damselfly condition (via lipid and muscle reduction) and fitness as urbanization increases.

Does juvenile hormone prompt oxidative stress in male damselflies?

In invertebrates, it has recently been reported that secondary sexual characteristics (SSCs) reflect the antioxidant defense of their bearers, but it is not known what physiological link maintains the honesty of those signals. Here, we used the damselfly Hetaerina americana to test whether juvenile hormone plays such a role. First, we analyzed whether oxidative damage is a real threat in natural damselfly populations by examining the accumulation of oxidized guanines as a function of age in males. Then, we injected paraquat (a pro-oxidant agent) and added the juvenile hormone analog methoprene (JHa) to the experimental group and the JHa vehicle (acetone) to the control group, to determine whether JHa increases the levels of pro-oxidants and antioxidants. We found that DNA oxidation increased with age, and that levels of hydrogen peroxide and superoxide dismutase, but not catalase or glutathione, were elevated in the JHa group compared with the control group. We propose that juvenile hormone is a mediator of the relationship between SSCs and antioxidant capacity and, based on the literature, we know that JHa suppresses the immune response. We therefore suggest that juvenile hormone is a molecular mediator of the general health of males, which is reflected in their SSCs.

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.

Sexual signals reveal males’ oxidative stress defences: Testing this hypothesis in an invertebrate

Abstract The hypothesis that sexual traits reveal the oxidative stress resistance of their bearers has been widely tested in vertebrates but remains unexplored in invertebrates. Here, Hetaerina americana was used to test whether oxidative stress defences are advertised by male wing spot size and colour (a male sexual trait). To this end, we investigated (1) whether oxidative stress reduced survival, (2) whether wing spot size revealed males’ antioxidant defences and (3) how wing spot size and colour were affected by oxidative stress. We elevated oxidative stress by injecting adult males with paraquat (PQ, a compound that favours the production of free radicals) and then examined how this affected male survival and wing spot size. We then related the expression of wing spot size to indicators of oxidative stress—hydrogen peroxide (H2O2) and total antioxidant capacity (TAC), super oxide dismutase (SOD) and catalase (CAT)—in adult males (whose wing spot is fixed) injected with PQ. In teneral males, whose wing spots are still forming, we compared wing spot size, colour (red and yellow chroma) and brightness in individuals injected with PQ or water as a control. Oxidative stress reduced the survival of adult and teneral males. While the H2O2 and TAC markers of antioxidant defences were positively correlated with wing spot size, there was no correlation with CAT and a negative correlation with SOD. In teneral males, PQ increased the yellow chroma and brightness of wing spots, but did not affect spot size or red chroma. Our results highlight the importance of measuring different markers as indicators of male oxidative stress defences, and that the sexual signals of invertebrates may reveal the oxidative stress status of their bearers. A plain language summary is available for this article.

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.

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.

A Mismatch between the Perceived Fighting Signal and Fighting Ability Reveals Survival and Physiological Costs for Bearers

Signals of fighting indicate an animal's intention to attack and so they serve to prevent costly aggressive encounters. However, according to theory, a signal that is different in design (i.e. a novel signal) but that fails to inform fighting intentions will result in negative fitness consequences for the bearer. In the present study we used males of the territorial damselfly Hetaerina americana, which have a red wing spot during territory defense that has evolved as a signal of fighting ability. By producing a novel signal (covering the red spot with blue ink) in territory owners, we investigated: a) the behavioral responses by conspecific males; b) survival cost and c) three physiological mediators of impaired survival: muscular fat reserves, muscle mass and immune ability. We predicted that males with the novel signal would be attacked more often by conspecifics as the former would fail to convey fighting ability and intentions adequately. This will result in lower survival and physiological condition for the novel signal bearers. We found that, compared to control males (males whose red spot was not changed), experimental males had reduced survival, were less able to hold a territory, and had a reduced muscle mass. It seems that spot modified males were not able to effectively communicate their territory tenancy, which may explain why they lost their defended sites. Our results provide support for theoretical models that a novel signal that fails to informing fighting ability may lead to a fitness cost for bearers.

Effect of juvenile hormone on senescence in males with terminal investment

Senescence, a decline in survival and reproductive prospects with age, is controlled by hormones. In insects, juvenile hormone (JH) is involved in senescence with captive individuals, but its effect under natural conditions is unknown. We have addressed this gap by increasing JH levels in young and old wild males of the damselfly Hetaerina americana. We assessed survival in males that were treated with a JH analogue (methoprene), which is known to promote sexual activity, and an immune challenge, which is known to promote terminal investment in reproduction in the studied species. We replicated the same procedure in captivity (to control for environmental variation), where males were deprived of any activity or food. We expected old males to show the lowest survival after being treated with JH and immune-challenged, because the effect of terminal investment on senescence would be exacerbated by JH. However, this should be the case for wild animals, but not for captive animals, as the effects of JH and immune challenge should lead to an increase in high energetic-demanding activities only occurring in the wild. Old animals died sooner compared with young animals in both the wild and captivity, confirming that males are subject to senescence. In wild but not captive animals, JH decreased survival in young males and increased it in old males, confirming that JH is sensitive to the environment when shaping animal senescence. Immune challenge had no effect on survival, suggesting no effect of terminal investment on senescence. Additionally, contrary to the expected effects of terminal investment, with an immune challenge, recapture rates increased in young males and decreased in old males. Our results show that male senescence in the wild is mediated by JH and that terminal investment does not cause senescence. One explanation is that animals undergoing senescence and terminal investment modify their feeding behaviour to compensate for their physiological state.

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.

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.