Interspecific interactions influence contrasting spatial genetic structures in two closely related damselfly species.

Spatial genetic structure (SGS) is largely determined by colonization history, landscape and ecological characteristics of the species. Therefore, sympatric and ecologically similar species are expected to exhibit similar SGSs, potentially enabling prediction of the SGS of one species from that of another. On the other hand, due to interspecific interactions, ecologically similar species could have different SGSs. We explored the SGSs of the closely related Calopteryx splendens and Calopteryx virgo within Finland and related the genetic patterns to characteristics of the sampling localities. We observed different SGSs for the two species. Genetic differentiation even within short distances in C. splendens suggests genetic drift as an important driver. However, we also observed indication of previous gene flow (revealed by a negative relationship between genetic differentiation and increasing potential connectivity of the landscape). Interestingly, genetic diversity of C. splendens was negatively related to density of C. virgo, suggesting that interspecific interactions influence the SGS of C. splendens. In contrast, genetic differentiation between C. virgo subpopulations was low and only exhibited relationships with latitude, pointing to high gene flow, colonization history and range margin effects as the drivers of SGS. The different SGSs of the two ecologically similar species caution indirect inferences of SGS based on ecologically similar surrogate species.

Sex-related effects in the superhydrophobic properties of damselfly wings in young and old Calopteryx splendens.

Numerous sex-related morphological adaptations are connected to reproductive behavior in animals. For example, females of some insect species can submerge during oviposition, which may lead to sex-related adaptations in the hydrophobicity (water-repellency) due to specialization of certain morphological structures. On the other hand, ageing can cause changes in hydrophobicity of the surface, because the morphological structures can wear with age. Here, we investigated sex-and age-related differences in wing hydrophobicity and in morphology (spine density, wax cover characteristics, size of females' pseudopterostigma) potentially related to hydrophobicity of Calopteryx splendens damselflies. Hydrophobicity was measured with two methods, by measuring the contact angle (CA) between a wing and water droplet, and by dipping a wing into water and measuring forces needed to submerge, withdraw, and pull-out a wing from water. We found that C. splendens wings are superhydrophobic, having mean CAs of 161°. The only sex and age related difference in the hydrophobicity measurements was that young females had stronger amplitude of force fluctuations during withdrawal of wings from water than young males. This suggests that young females may form less uniform air pockets on their wings while submerged. From the morphological structures measured here, the only sex related finding was that old females had denser spine cover than young females in their wing veins. The difference may be explained by better survival of females with denser spine cover. The most important morphological character that predicted superhydrophobicity was the prevalence of long wax rods on wing veins. In addition, female pseudopterostigma area (a trait present only in females) was negatively related to pull-out force, suggesting that large pseudopterostigmas might help females to emerge from water following oviposition. The subtle sex-related differences in hydrophobicity could be explained by the fact that both sexes must resist rain, and males are occasionally in contact with water.

Do allopatric male Calopteryx virgo damselflies learn species recognition?

There is a growing amount of empirical evidence that premating reproductive isolation of two closely related species can be reinforced by natural selection arising from avoidance of maladaptive hybridization. However, as an alternative for this popular reinforcement theory, it has been suggested that learning to prefer conspecifics or to discriminate heterospecifics could cause a similar pattern of reinforced premating isolation, but this possibility is much less studied. Here, we report results of a field experiment in which we examined (i) whether allopatric Calopteryx virgo damselfly males that have not encountered heterospecific females of the congener C. splendens initially show discrimination, and (ii) whether C. virgo males learn to discriminate heterospecifics or learn to associate with conspecifics during repeated experimental presentation of females. Our experiment revealed that there was a statistically nonsignificant tendency for C. virgo males to show initial discrimination against heterospecific females but because we did not use sexually naïve individuals in our experiment, we were not able to separate the effect of innate or associative learning. More importantly, however, our study revealed that species discrimination might be further strengthened by learning, especially so that C. virgo males increase their association with conspecific females during repeated presentation trials. The role of learning to discriminate C. splendens females was less clear. We conclude that learning might play a role in species recognition also when individuals are not naïve but have already encountered potential conspecific mates.

Effects of cuticle structure and crystalline wax coverage on the coloration in young and old males of Calopteryx splendens and Calopteryx virgo.

Male secondary sexual characters, such as color patterns, are often investigated at the macroscale level. However, micro- and nanoscale levels of morphological investigations may reveal functional features responsible for a particular coloration, thus providing more information, e.g., about the condition dependence of male sexual characters. The aim of this paper was to investigate cuticle color and its structure in males of two congeneric damselfly species, Calopteryx splendens and Calopteryx virgo, and reveal possible color changes with age. According to spectrometer measurements, C. splendens males were bluer and had a greater saturation of blue in their abdomen than C. virgo males, which were, in turn, greener and had more green saturation. Although the two species differed in the number of structural layers and the spacing of the layers, it seems that intactness of the wax crystals covering the epicuticle was most often the morphological trait which was related to the color parameters measured from males' cuticles. The effect of the crystalline wax coverage on cuticle color was also confirmed by removing the wax using chloroform: after the treatment, the hue was bluer, the cuticle had a greater brightness and greater blue saturation, but less green saturation. Age differences influencing the color and structure of the cuticle were also observed: older males had more blue and green saturation and had more intact wax coverage than did younger males. Although multilayer reflection should be responsible for the iridescent color of males, our results suggest that wax coverage plays an important role in the color tuning of the male cuticle. This may have a considerable signal function, indicating the males' viability to competing males or to females.