Widespread Wolbachia infection in an insular radiation of damselflies (Odonata, Coenagrionidae).

Wolbachia is one of the most common endosymbionts found infecting arthropods. Theory predicts symbionts like Wolbachia will be more common in species radiations, as host shift events occur with greatest frequency between closely related species. Further, the presence of Wolbachia itself may engender reproductive isolation, and promote speciation of their hosts. Here we screened 178 individuals belonging to 30 species of the damselfly genera Nesobasis and Melanesobasis - species radiations endemic to the Fiji archipelago in the South Pacific - for Wolbachia, using multilocus sequence typing to characterize bacterial strains. Incidence of Wolbachia was 71% in Nesobasis and 40% in Melanesobasis, and prevalence was also high, with an average of 88% in the Nesobasis species screened. We identified a total of 25 Wolbachia strains, belonging to supergroups A, B and F, with some epidemic strains present in multiple species. The occurrence of Wolbachia in both males and females, and the similar global prevalence found in both sexes rules out any strong effect of Wolbachia on the primary sex-ratio, but are compatible with the phenotype of cytoplasmic incompatibility. Nesobasis has higher species richness than most endemic island damselfly genera, and we discuss the potential for endosymbiont-mediated speciation within this group.

Female Colour Polymorphism and Unique Reproductive Behaviour in Polythore Damselflies (Zygoptera: Polythoridae).

We studied Polythore damselflies by mark-recapture techniques in the Jatun Sacha Biological Reserve (Ecuador) for a period of 48 days in October-December 2014. Three species were found: Polythore mutata (MacLachlan) was the commonest species (111 individuals marked), Polythore derivata (MacLachlan) was rare (24 individuals) and Polythore concinna (MacLachlan) occasional (four individuals). In P. mutata, we found two phenotypes amongst females, one of them with a white band on the wings, very similar in colouration to the conspecific male (androchrome), and the other with an amber band (gynochrome). The recapture of marked females indicates that both phenotypes are maintained since emergence to maturation and are not age-related (i.e. polymorphism). Androchromes represent 40% of females observed. The colour of the wing band showed an age-dependent change in size with opposite trends between sexes, increasing in males and decreasing in females. Males and females were observed to return to the same forest locations in different days. Courtships and ovipositions involving androchrome females were not observed. No matings were observed in any morph. In contrast, we recorded two consecutive matings of one female P. derivata. We found that Polythore males grasp the mesothorax of females during mating instead of the prothorax as in other Zygoptera. We discuss the rarity of reproductive behaviour in this genus and how female morphs might be maintained.

Genetic divergence predicts reproductive isolation in damselflies.

Reproductive isolation is the defining characteristic of a biological species, and a common, but often untested prediction is a positive correlation between reproductive isolation and genetic divergence. Here, we test for this correlation in odonates, an order characterized by strong sexual selection. First, we measure reproductive isolation and genetic divergence in eight damselfly genera (30 species pairs) and test for a positive correlation. Second, we estimate the genetic threshold preventing hybrid formation and empirically test this threshold using wild populations of species within the Ischnura genus. Our results indicate a positive and strong correlation between reproductive isolation and genetic distance using both mitochondrial and nuclear genes cytochrome oxidase II (COII: r = 0.781 and 18S-28S: r = 0.658). Hybridization thresholds range from -0.43 to 1.78% for COII and -0.052-0.71% for 18S-28S, and both F1 -hybrids and backcrosses were detected in wild populations of two pairs of Ischnura species with overlapping thresholds. Our study suggests that threshold values are suitable to identify species prone to hybridization and that positive isolation-divergence relationships are taxonomically widespread.

Population genetic structure of sexual and parthenogenetic damselflies inferred from mitochondrial and nuclear markers.

It has been postulated that obligate asexual lineages may persist in the long term if they escape from negative interactions with either sexual lineages or biological enemies; and thus, parthenogenetic populations will be more likely to occur in places that are difficult for sexuals to colonize, or those in which biological interactions are rare, such as islands or island-like habitats. Ischnura hastata is the only known example of natural parthenogenesis within the insect order Odonata, and it represents also a typical example of geographic parthenogenesis, as sexual populations are widely distributed in North America, whereas parthenogenetic populations of this species have only been found at the Azores archipelago. In order to gain insight in the origin and distribution of parthenogenetic I. hastata lineages, we have used microsatellites, mitochondrial and nuclear DNA sequence data, to examine the population genetic structure of this species over a wide geographic area. Our results suggest that sexual populations of I. hastata in North America conform to a large subdivided population that has gone through a recent spatial expansion. A recent single long distance dispersal event, followed by a demographic expansion, is the most parsimonious hypothesis explaining the origin of the parthenogenetic population of this species in the Azores islands.

Genetic drift and rapid evolution of viviparity in insular fire salamanders (Salamandra salamandra).

Continental islands offer an excellent opportunity to investigate adaptive processes and to time microevolutionary changes that precede macroevolutionary events. We performed a population genetic study of the fire salamander (Salamandra salamandra), a species that displays unique intraspecific diversity of reproductive strategies, to address the microevolutionary processes leading to phenotypic and genetic differentiation of island, coastal and interior populations. We used eight microsatellite markers to estimate genetic diversity, population structure and demographic parameters in viviparous insular populations and ovoviviparous coastal and interior populations. Our results show considerable genetic differentiation (F(ST) range: 0.06-0.27), and no clear signs of gene flow among populations, except between the large and admixed interior populations. We find no support for island colonization by rafting or intentional/accidental anthropogenic introductions, indicating that rising sea levels were responsible for isolation of the island populations approximately 9000 years ago. Our study provides evidence of rapid genetic differentiation between island and coastal populations, and rapid evolution of viviparity driven by climatic selective pressures on island populations, geographic isolation with genetic drift, or a combination of these factors. Studies of these viviparous island populations in early stages of divergence help us better understand the microevolutionary processes involved in rapid phenotypic shifts.

The influence of stochastic and selective forces in the population divergence of female colour polymorphism in damselflies of the genus Ischnura.

Disentangling the relative importance and potential interactions of selection and genetic drift in driving phenotypic divergence of species is a classical research topic in population genetics and evolutionary biology. Here, we evaluate the role of stochastic and selective forces on population divergence of a colour polymorphism in seven damselfly species of the genus Ischnura, with a particular focus on I. elegans and I. graellsii. Colour-morph frequencies in Spanish I. elegans populations varied greatly, even at a local scale, whereas more similar frequencies were found among populations in eastern Europe. In contrast, I. graellsii and the other five Ischnura species showed little variation in colour-morph frequencies between populations. F(ST)-outlier analyses revealed that the colour locus deviated strongly from neutral expectations in Spanish populations of I. elegans, contrasting the pattern found in eastern European populations, and in I. graellsii, where no such discrepancy between morph divergence and neutral divergence could be detected. This suggests that divergent selection has been operating on the colour locus in Spanish populations of I. elegans, whereas processes such as genetic drift, possibly in combination with other forms of selection (such as negative frequency-dependent selection), appear to have been present in other regions, such as eastern Europe. Overall, the results indicate that both selective and stochastic processes operate on these colour polymorphisms, and suggest that the relative importance of factors varies between geographical regions.

Larval aquatic and terrestrial mites infesting parthenogenetic Ischnura hastata (Odonata: Coenagrionidae) from the Azores islands.

We report here the prevalence of parasitism by water mites (Arrenurus sp.) and terrestrial mites (Leptus killingtoni) on parthenogenetic Ischnura hastata (Odonata: Coenagrionidae) from the Azores islands. Leptus killingtoni was only found on the island of Pico, and the prevalence of infestation was highly variable among the different ponds studied, ranging from 0 to 41%. Leptus killingtoni was observed on three of the four odonate species from the archipelago: I. hastata, I. pumilio, and Sympetrum fonscolombii, all of them new hosts for this species. Aquatic mites have been found parasitizing I. hastata females on the island of São Miguel. The prevalence of mite parasitism by Arrenurus sp. on I. hastata was very low, ranging from 12% (2003) to 1% (2008), and in most of the studied ponds, no mites were found attached to females. Although I. hastata coexists with a sexual congener species in the Azores (I. pumilio), they are syntopic in only a small fraction of ponds. Therefore, a comparison between I. hastata and I. pumilio was insufficient to test the predictions of the Red Queen Hypothesis, and further research on parasitism rates in both species needs to be done. In any case, the low prevalence of mite parasitism found in the Azores, coupled with the fact that most of the populations in the archipelago are almost free from competitors and predators, could explain the persistence of these I. hastata parthenogenetic populations, despite their low levels of genetic variation.

A comparative analysis of senescence in adult damselflies and dragonflies (Odonata).

Any population whose members are subject to extrinsic mortality should exhibit an increase in mortality with age. Nevertheless, the prevailing opinion is that populations of adult damselflies and dragonflies do not exhibit such senescence. Here, we challenge this contention by fitting a range of demographic models to the data on which these earlier conclusions were based. We show that a model with an exponential increase in age-related mortality (Gompertz) generally provides a more parsimonious fit than alternative models including age-independent mortality, indicating that many odonates do indeed senesce. Controlling for phylogeny, a comparison of the daily mortality of 35 odonate species indicates that although male and female mortalities are positively correlated, mortality tends to be higher in males of those species that exhibit territoriality. Hence, we show for the first time that territoriality may impose a survivorship cost on males, once the underlying phylogenetic relationships are accounted for.

Three sisters in the same dress: cryptic speciation in African odonates.

The discovery of cryptic species (i.e. two or more distinct but morphologically undistinguishable species) has grown exponentially in the last two decades, due mainly to the increasing availability of DNA sequences. This suggests that hidden in the known species, many of which have been described based solely on morphological information, there might be a high number of species waiting to be discovered. In this issue Damm et al. (2010) use a combination of genetic, morphological and ecological evidence to identify the first cryptic species complex found within dragonflies (insect order Odonata). Their findings add more evidence for the importance of combining information from different disciplines to new species' discovery (DeSalle et al. 2005).

Empirical evidence of senescence in adult damselflies (Odonata: Zygoptera).

1. Age-dependent increases in mortality have been documented in a variety of species of insect under laboratory conditions. However, while strong statistical evidence has been presented for senescence in vertebrate populations in the wild, we know little about the rate and shape of senescence in wild populations of insects. 2. Odonates (damselflies and dragonflies) provide excellent candidate species for evaluating demographic senescence as they are large enough to be marked individually and they are easily re-sighted without recapture. The prevailing opinion - based entirely on qualitative examination of the declines in log numbers alive with time since marking - is that odonates exhibit age-independent daily survivorship. 3. Here, we examine mark-recapture data on the Azure Damselfly Coenagrion puella over two consecutive seasons. For the first time, we evaluate and compare the fit of quantitative models that not only account for weather-dependent daily variation in daily re-sighting rates, but also age-dependent variation in daily survivorship. 4. Models with age-dependent declines in daily survivorship provide a more parsimonious explanation for the data than similar models without these age-dependent effects. In general, models in which mortality increases in an exponential (Gompertz) fashion explain the mark-recapture sequences more efficiently than a range of alternative models, including those in which mortality increases as a power function (Weibull) or reaches a plateau (logistic). These results are indicative of a general senescent decline in physiological functioning, which is particularly marked after 15 days as a mature adult. 5. Weather (temperature, sun and precipitation) and initial mite load influenced the probability of daily re-sighting. Weather and mite load also influenced daily survivorship, but their effects differed between seasons. 6. Overall, fitting models with age as an explicit covariate demonstrates that odonates do indeed senesce. This contradicts previously held assumptions that Odonata do not exhibit age-dependent survivorship in the wild.

Thelytokous parthenogenesis in the damselfly Ischnura hastata (Odonata, Coenagrionidae): genetic mechanisms and lack of bacterial infection.

Thelytokous parthenogenesis, the production of female-only offspring from unfertilized eggs, has been described in all the insect orders, but is a rare phenomenon in the Odonata (dragonflies and damselflies). The only-known case of parthenogenesis in this group is the North American damselfly species Ischnura hastata, which has parthenogenetic populations in the Azores Islands. Here, we present for the first time the results of laboratory rearing, which showed parthenogenetic reproduction in the Azorean I. hastata populations. In an attempt to understand how parthenogenesis could have evolved in this species, we first determined the genetic mode of parthenogenesis by analysing the genotype of parthenogenetic females and their offspring at three polymorphic microsatellite loci. In addition, we used polymerase chain reaction amplification to test whether parthenogenesis in I. hastata could be bacterially induced. Our data indicate that thelytoky is achieved through an (at least functionally) apomictic mechanism and that parthenogenesis is not caused by endosymbionts. Finally, we discuss possible routes to parthenogenetic reproduction, as well as the evolutionary implications of this type of parthenogenesis.

Postmating sexual selection: allopatric evolution of sperm competition mechanisms and genital morphology in calopterygid damselflies (Insecta: Odonata).

Postmating sexual selection theory predicts that in allopatry reproductive traits diverge rapidly and that the resulting differentiation in these traits may lead to restrictions to gene flow between populations and, eventually, reproductive isolation. In this paper we explore the potential for this premise in a group of damselflies of the family Calopterygidae, in which postmating sexual mechanisms are especially well understood. Particularly, we tested if in allopatric populations the sperm competition mechanisms and genitalic traits involved in these mechanisms have indeed diverged as sexual selection theory predicts. We did so in two different steps. First, we compared the sperm competition mechanisms of two allopatric populations of Calopteryx haemorrhoidalis (one Italian population studied here and one Spanish population previously studied). Our results indicate that in both populations males are able to displace spermathecal sperm, but the mechanism used for sperm removal between both populations is strikingly different. In the Spanish population males seem to empty the spermathecae by stimulating females, whereas in the Italian population males physically remove sperm from the spermathecae. Both populations also exhibit differences in genital morphometry that explain the use of different mechanisms: the male lateral processes are narrower than the spermathecal ducts in the Italian population, which is the reverse in the Spanish population. The estimated degree of phenotypic differentiation between these populations based on the genitalic traits involved in sperm removal was much greater than the differentiation based on a set of other seven morphological variables, suggesting that strong directional postmating sexual selection is indeed the main evolutionary force behind the reproductive differentiation between the studied populations. In a second step, we examined if a similar pattern in genital morphometry emerge in allopatric populations of this and other three species of the same family (Calopteryx splendens, C. virgo and Hetaerina cruentata). Our results suggest that there is geographic variation in the sperm competition mechanisms in all four studied species. Furthermore, genitalic morphology was significantly divergent between populations within species even when different populations were using the same copulatory mechanism. These results can be explained by probable local coadaptation processes that have given rise to an ability or inability to reach and displace spermathecal sperm in different populations. This set of results provides the first direct evidence of intraspecific evolution of genitalic traits shaped by postmating sexual selection.

Copulation duration and fertilization success in a damselfly: an example of cryptic female choice?

Copulation duration is highly variable (0.5-3 h) in the damselfly, Ceriagrion tenellum (Coenagrionidae). Using laboratory experiments, we tested four adaptive hypotheses to explain this variation: the effect of time constraints, in-copula mate guarding, sperm displacement and cryptic female choice. Copulation duration was negatively correlated with time of day, as predicted by the first two hypotheses, and positively correlated with male density, as predicted by the mate-guarding hypothesis. Males prolonged copulation in response to the volume of sperm stored by females, suggesting they were able to detect and quantify the amount of sperm stored. This behaviour is not explained by mate guarding or time constraint effects. Males removed all the sperm from the bursa copulatrix in just 10 min. Our results also suggest that, because the duct is too narrow to allow male genitalia to enter, males do not remove spermathecal sperm. Therefore, direct sperm removal could not explain long copulations. Prolonged copulations could also have evolved as a result of cryptic female choice if they increase male fertilization success by female-mediated processes. Our results support this idea: male fertilization success was greater after long copulations. Apparently, male copulatory behaviour elicits female responses that increase male fertilization success. Copyright 2000 The Association for the Study of Animal Behaviour.

Molecular evidence for selection on female color polymorphism in the damselfly Ischnura graellsii.

The significance of female color polymorphism in Odonata remains controversial despite many field studies. The importance of random factors (founder effects, genetic drift and migration) versus selective forces for the maintenance of this polymorphism is still discussed. In this study, we specifically test whether the female color polymorphism of Ischnura graellsii (Odonata, Coenagrionidae) is under selection in the wild. We compared the degree of genetic differentiation based on RAPD markers (assumed to be neutral) with the degree of differentiation based on color alleles. Weir and Cockerham's theta values showed a significant degree of population differentiation for both sets of loci (RAPD and color alleles) but the estimated degree of population differentiation (theta) was significantly greater for the set of RAPD loci. This result shows that some sort of selection contributes to the maintenance of similar color morph frequencies across the studied populations. Our results combined with those of previous field studies suggest that at least in some I. graellsii populations, density-dependent mechanisms might help to prevent the loss of this polymorphism but cannot explain the similarity in morph frequencies among populations.