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Petaluridae (Odonata: Anisoptera) is a relict dragonfly family, having diverged from its sister family in the Jurassic, of eleven species that are notable among odonates (dragonflies and damselflies) for their exclusive use of fen and bog habitats, their burrowing behavior as nymphs, large body size as adults, and extended lifespans. To date, several nodes within this family remain unresolved, limiting the study of the evolution of this peculiar family. Using an anchored hybrid enrichment dataset of over 900 loci we reconstructed the species tree of Petaluridae. To estimate the temporal origin of the genera within this family, we used a set of well-vetted fossils and a relaxed molecular clock model in a divergence time estimation analysis. We estimate that Petaluridae originated in the early Cretaceous and confirm the existence of monophyletic Gondwanan and Laurasian clades within the family. Our relaxed molecular clock analysis estimated that these clades diverged from their MRCA approximately 160 mya. Extant lineages within this family were identified to have persisted from 6 (Uropetala) to 120 million years (Phenes). Our biogeographical analyses focusing on a set of key regions suggest that divergence within Petaluridae is largely correlated with continental drift, the exposure of land bridges, and the development of mountain ranges. Our results support the hypothesis that species within Petaluridae have persisted for tens of millions of years, with little fossil evidence to suggest widespread extinction in the family, despite optimal conditions for the fossilization of nymphs. Petaluridae appear to be a rare example of habitat specialists that have persisted for tens of millions of years., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Reproductive Character Displacement (RCD) refers to the phenomenon of greater differences in reproductive characters between two species when they occur in sympatry compared to when they occur in allopatry to prevent maladaptive hybridization. We explored whether reinforcement of a mechanical barrier involved in the first contact point between male and female genital traits during copulation in the cross between Ischnura graellsii males and Ischnura elegans females has led to RCD, and whether it supports the lock-and-key hypothesis of genital evolution. We employed geometric morphometrics to analyze the shape and size of male and female genital traits, controlling for environmental and geographic factors. Consistent with an increase in mechanical isolation via reinforcement, we detected larger divergence in genital traits between the species in sympatry than in allopatry, and also stronger signal in females than in males. In the Northwest (NW) hybrid zone, we detected RCD in I. graellsii males and I. elegans females, while in the Northcentral (NC) hybrid zone we detected RCD only in I. elegans females and I. elegans males. The detection of RCD in both sexes of I. elegans was consistent with the lock-and-key hypothesis of genital evolution via female choice for conspecific males in this species. Our study highlights the importance of using geometric morphometrics to deal with the complexity of female reproductive structures while controlling for environmental and geographic factors to investigate RCD. This study contributes valuable insights into the dynamics of reproductive isolation mechanisms and genital coevolution., (© 2024. The Author(s), under exclusive licence to The Genetics Society.)

The processes responsible for the formation of Earth's most conspicuous diversity pattern, the latitudinal diversity gradient (LDG), remain unexplored for many clades in the Tree of Life. Here, we present a densely sampled and dated molecular phylogeny for the most speciose clade of damselflies worldwide (Odonata: Coenagrionoidea) and investigate the role of time, macroevolutionary processes, and biome-shift dynamics in shaping the LDG in this ancient insect superfamily. We used process-based biogeographic models to jointly infer ancestral ranges and speciation times and to characterize within-biome dispersal and biome-shift dynamics across the cosmopolitan distribution of Coenagrionoidea. We also investigated temporal and biome-dependent variation in diversification rates. Our results uncover a tropical origin of pond damselflies and featherlegs ~105 Ma, while highlighting the uncertainty of ancestral ranges within the tropics in deep time. Even though diversification rates have declined since the origin of this clade, global climate change and biome-shifts have slowly increased diversity in warm- and cold-temperate areas, where lineage turnover rates have been relatively higher. This study underscores the importance of biogeographic origin and time to diversify as important drivers of the LDG in pond damselflies and their relatives, while diversification dynamics have instead resulted in the formation of ephemeral species in temperate regions. Biome-shifts, although limited by tropical niche conservatism, have been the main factor reducing the steepness of the LDG in the last 30 Myr. With ongoing climate change and increasing northward range expansions of many damselfly taxa, the LDG may become less pronounced. Our results support recent calls to unify biogeographic and macroevolutionary approaches to improve our understanding of how latitudinal diversity gradients are formed and why they vary across time and among taxa., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Systematic Biologists.)

Winter diapause consists of cessation of development that allows individuals to survive unfavourable conditions. Winter diapause may bear various costs and questions have been raised about the evolutionary mechanisms maintaining facultative diapause. Here, we explored to what extent a facultative winter diapause affects life-history traits and the transcriptome in the damselfly Ischnura elegans, and whether these effects were latitude-specific. We collected adult females at central and high latitudes and raised their larvae in growth chambers. Larvae were split into a non-diapausing and post-winter (diapausing) cohort, were phenotyped and collected for a gene expression analysis. At the phenotypic level, we found no difference in survival between the two cohorts, and the post-winter cohort was larger and heavier than the non-winter cohort. These effects were mostly independent of the latitude of origin. At the transcriptomic level, wintering affected gene expression with a small fraction of genes significantly overlapping across latitudes, especially those related to morphogenesis. In conclusion, we found clear effects of diapause on the phenotype but little evidence for latitudinal-specific effects of diapause. Our results showed a shared transcriptomic basis underpinning diapause demonstrated, here, at the intraspecific level and supported the idea of evolutionary convergence of the response to diapause across organisms., (© 2024. The Author(s).)

Despite is known to have widespread distribution and the most active species of the family Chlorocyphidae, the molecular data of Rhinocypha fenestrella (Rambur, 1842) are relatively scarce. The present study is the first that examined the genetic diversity and phylogeographic pattern of the peacock jewel-damselfly R. fenestrella by sequencing the cytochrome C oxidase I (cox1) and 16S rRNA gene regions from 147 individuals representing eight populations in Malaysia. A total of 26 and 10 unique haplotypes were revealed by the cox1 and 16S rRNA genes, respectively, and 32 haplotypes were recovered by the concatenated sequences of cox1+16S. Analyses indicated that haplotype AB2 was the most frequent and the most widespread haplotype in Malaysia while haplotype AB1 was suggested as the common ancestor haplotype of the R. fenestrella that may arose from the Negeri Sembilan as discovered from cox1+16S haplotype network analysis. Overall haplotype and nucleotide diversities of the concatenated sequences were Hd = 0.8937 and Pi = 0.0028, respectively, with great genetic differentiation (FST = 0.6387) and low gene flow (Nm = 0.14). Population from Pahang presented the highest genetic diversity (Hd = 0.8889, Pi = 0.0022, Nh = 9), whereas Kedah population demonstrated the lowest diversity (Hd = 0.2842, Pi = 0.0003, Nh = 4). The concatenated sequences of cox1+16S showed genetic divergence ranging from 0.09% to 0.97%, whereas the genetic divergence for cox1 and 16S rRNA genes were 0.16% to 1.63% and 0.01% to 0.75% respectively. This study provides for the first-time insights on the intraspecific genetic diversity, phylogeographic pattern and ancestral haplotype of Rhinocypha fenestrella. The understanding of molecular data especially phylogeographic pattern can enhance the knowledge about insect origin, their diversity, and capability to disperse in particular environments., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Noorhidayah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© The American Genetic Association. 2023.)

Background: Describing geographical variation in morphology of organisms in combination with data on genetic differentiation and biogeography can provide important information on how natural selection shapes such variation. Here we study genetic structure using ddRAD seq and wing shape variation using geometric morphometrics in 14 populations of the damselfly Lestes sponsa along its latitudinal range in Europe., Results: The genetic analysis showed a significant, yet relatively weak population structure with high genetic heterozygosity and low inbreeding coefficients, indicating that neutral processes contributed very little to the observed wing shape differences. The genetic analysis also showed that some regions of the genome (about 10%) are putatively shaped by selection. The phylogenetic analysis showed that the Spanish and French populations were the ancestral ones with northern Swedish and Finnish populations being the most derived ones. We found that wing shape differed significantly among populations and showed a significant quadratic (but weak) relationship with latitude. This latitudinal relationship was largely attributed to allometric effects of wing size, but non-allometric variation also explained a portion of this relationship. However, wing shape showed no phylogenetic signal suggesting that lineage-specific variation did not contribute to the variation along the latitudinal gradient. In contrast, wing size, which is correlated with body size in L. sponsa, had a strong negative correlation with latitude., Conclusion: Our results suggest a relatively weak population structure among the sampled populations across Europe, but a clear differentiation between south and north populations. The observed geographic phenotypic variation in wing shape may have been affected by different local selection pressures or environmental effects., (© 2024. The Author(s).)

Sex-limited morphs can provide profound insights into the evolution and genomic architecture of complex phenotypes. Inter-sexual mimicry is one particular type of sex-limited polymorphism in which a novel morph resembles the opposite sex. While inter-sexual mimics are known in both sexes and a diverse range of animals, their evolutionary origin is poorly understood. Here, we investigated the genomic basis of female-limited morphs and male mimicry in the common bluetail damselfly. Differential gene expression between morphs has been documented in damselflies, but no causal locus has been previously identified. We found that male mimicry originated in an ancestrally sexually dimorphic lineage in association with multiple structural changes, probably driven by transposable element activity. These changes resulted in ~900 kb of novel genomic content that is partly shared by male mimics in a close relative, indicating that male mimicry is a trans-species polymorphism. More recently, a third morph originated following the translocation of part of the male-mimicry sequence into a genomic position ~3.5 mb apart. We provide evidence of balancing selection maintaining male mimicry, in line with previous field population studies. Our results underscore how structural variants affecting a handful of potentially regulatory genes and morph-specific genes can give rise to novel and complex phenotypic polymorphisms., (© 2023. The Author(s).)

Using recently published chromosome-length genome assemblies of two damselfly species, Ischnura elegans and Platycnemis pennipes, and two dragonfly species, Pantala flavescens and Tanypteryx hageni, we demonstrate that the autosomes of Odonata have undergone few fission, fusion, or inversion events, despite 250 million years of separation. In the four genomes discussed here, our results show that all autosomes have a clear ortholog in the ancestral karyotype. Despite this clear chromosomal orthology, we demonstrate that different factors, including concentration of repeat dynamics, GC content, relative position on the chromosome, and the relative proportion of coding sequence all influence the density of syntenic blocks across chromosomes. However, these factors do not interact to influence synteny the same way in any two pairs of species, nor is any one factor retained in all four species. Furthermore, it was previously unknown whether the micro-chromosomes in Odonata are descended from one ancestral chromosome. Despite structural rearrangements, our evidence suggests that the micro-chromosomes in the sampled Odonata do indeed descend from an ancestral chromosome, and that the micro-chromosome in P. flavescens was lost through fusion with autosomes., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Ischnura senegalensis Rambur, 1842 is among the most widespread damselfly species in the world. Unlike dragonflies with strong migration abilities, I. senegalensis have limited dispersing abilities. Gene flow among I. senegalensis populations may be greatly influenced by anthropogenic disturbance, fragmented suitable habitats, sea straits, or even global warming. In this study, to investigate the genetic diversity of I. senegalensis populations, we sequenced and collected 498 cytochrome oxidase I sequences across the Old World. Haplotype network analysis showed 51 haplotypes and I. senegalensis could be grouped into four regions (Afrotropical region, Oriental region, main Islands of Japan, and the Ryukyu Islands), each of which contains different dominant haplotypes. Based on molecular variance analysis, we found that populations from the Afrotropical region have quite a low gene flow with the Asian populations (except Yemen). Furthermore, rice cultivation may aid the dispersion of I. senegalensis in the oriental region. Populations from the Ryukyu Islands show the highest genetic diversity, which may be due to the geological separation among islands. Our results prove that I. senegalensis has great genetic diversity among different populations across the world., (© 2023 Wiley Periodicals LLC.)

South Pacific islands provide an ideal study system to explore patterns of speciation, specifically examining the role of dispersal versus vicariance. Dispersal is often the suggested mechanism of diversification in the South Pacific, specifically among remote island chains. Here, we provide a phylogeny of several related genera of Coenagrionidae (Odonata: Zygoptera) from the South Pacific, based on five molecular loci, in order to examine patterns of speciation in the region. We used the endemic damselfly genera Nesobasis, Nikoulabasis, and Vanuatubasis found across both Fiji and Vanuatu. Knowledge of the geologic history of the region was used to inform our understanding of the evolution of these genera. Both archipelagos used to be part of the Vitiaz arc which spanned from the Solomon Islands to Tonga and began to break apart 10-12 Ma. Results of our divergence-time estimations and biogeographic reconstructions support that the breakup of this arc acted as a significant vicariance event in the evolution of these taxa. Specifically, it led to the extant generic diversity seen in these damselflies. We find that within the archipelago of Vanuatu, that Espiritu Santo served as an important source for dispersal to other islands with Malekula acting as a stepping stone to Efate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Odonata is an order of insects that comprises ∼6500 species. They are among the earliest flying insects, and one of the first diverging lineages in the Pterygota. Odonate evolution has been a topic of research for over 100 years, with studies focusing primarily on their flight behavior, color, vision, and aquatic juvenile lifestyles. Recent genomics studies have provided new interpretations about the evolution of these traits. In this paper, we look at how high-throughput sequence data (i.e. subgenomic and genomic data) have been used to answer long-standing questions in Odonata ranging from evolutionary relationships to vision evolution to flight behavior. Additionally, we evaluate these data at multiple taxonomic levels (i.e. ordinal, familial, generic, and population) and provide comparative analysis of genomes across Odonata, identifying features of these new data. Last, we discuss the next two years of Odonata genomic study, with context about what questions are currently being tackled., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© The American Genetic Association. 2023.)

We present a chromosome-length genome assembly and annotation of the Black Petaltail dragonfly (Tanypteryx hageni). This habitat specialist diverged from its sister species over 70 million years ago, and separated from the most closely related Odonata with a reference genome 150 million years ago. Using PacBio HiFi reads and Hi-C data for scaffolding we produce one of the most high-quality Odonata genomes to date. A scaffold N50 of 206.6 Mb and a single copy BUSCO score of 96.2% indicate high contiguity and completeness., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The relationship between genetic differentiation and phenotypic plasticity can provide information on whether plasticity generally facilitates or hinders adaptation to environmental change. Here, we studied wing shape variation in a damselfly (Lestes sponsa) across a latitudinal gradient in Europe that differed in time constraints mediated by photoperiod and temperature. We reared damselflies from northern and southern populations in the laboratory using a reciprocal transplant experiment that simulated time-constrained (i.e. northern) and unconstrained (southern) photoperiods and temperatures. After emergence, adult wing shape was analysed using geometric morphometrics. Wings from individuals in the northern and southern populations differed significantly in shape when animals were reared in their respective native environment. Comparing wing shape across environments, we found evidence for phenotypic plasticity in wing shape, and this response differed across populations (i.e. G × E interactions). This interaction was driven by a stronger plastic response by individuals from the northern population and differences in the direction of plastic wing shape changes among populations. The alignment between genetic and plastic responses depended on the specific combination of population and rearing environment. For example, there was an alignment between plasticity and genetic differentiation under time-constrained, but not under non-time-constrained conditions for forewings. We thus find mixed support for the hypothesis that environmental plasticity and genetic population differentiation are aligned. Furthermore, although our laboratory treatments mimicked the natural climatic conditions at northern and southern latitudes, the effects of population differences on wing shape were two to four times stronger than plastic effects. We discuss our results in terms of time constraints and the possibility that natural and sexual selection is acting differently on fore- and hindwings., (© 2022 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.)

Contemporary hybrid zones act as natural laboratories for the investigation of species boundaries and may shed light on the little understood roles of sex chromosomes in species divergence. Sex chromosomes are considered to function as a hotspot of genetic divergence between species; indicated by less genomic introgression compared to autosomes during hybridization. Moreover, they are thought to contribute to Haldane's rule, which states that hybrids of the heterogametic sex are more likely to be inviable or sterile. To test these hypotheses, we used contemporary hybrid zones of Ischnura elegans , a damselfly species that has been expanding its range into the northern and western regions of Spain, leading to chronic hybridization with its sister species Ischnura graellsii . We analysed genome-wide SNPs in the Spanish I. elegans and I. graellsii hybrid zone and found (i) that the X chromosome shows less genomic introgression compared to autosomes, and (ii) that males are underrepresented among admixed individuals, as predicted by Haldane's rule. This is the first study in Odonata that suggests a role of the X chromosome in reproductive isolation. Moreover, our data add to the few studies on species with X0 sex determination system and contradict the hypothesis that the absence of a Y chromosome causes exceptions to Haldane's rule.

Among the oldest winged insects, odonates are a monophyletic order that have become important models for ecological studies because of their highly diverse reproductive behaviors and their role as top predators and bioindicators. However, knowledge on evolutionary relationships within the order is still scarce compared to other taxa, and this situation is even more complicated in areas with high biodiversity, such as in the Amazon. Here, we sought to identify knowledge gaps on Amazonian Odonata regarding three main aspects: (i) how the inclusion of Amazonian taxa affects our interpretation of the evolutionary relationships of Zygoptera and Anisoptera; (ii) the position of Amazonian taxa in the existing supertree of the Odonata; (iii) dating evolutionary divergence between nodes using fossil records; (iv) assessing whether more species-rich basins (e.g., Amazon basin) have a larger phylogenetic gap when compared to basins with lower richness in South and Central America; and (v) in the light of our knowledge, we discuss diversification patterns found in the most predominant clades of Amazonian taxa. We built a supertree from currently available phylogenetic information of Odonata. The results show that there is no genetic information for 85% (n: 503) of the Amazonian species and that family level relationships are unknown for 17 genera. After compiling the data, we observed that clades belonging to Neotropical lineages are the most poorly resolved, with large polytomies. This problem was identified in many Anisoptera genera, such as Macrothemis, Dasythemis, Elasmothemis, and Erythrodiplax. Our results also suggest that not always the richest basins have the greatest phylogenetic gaps. As expected, we found important gaps in the existing Odonata phylogenies, especially in clades that include Amazonian representatives, that are also those less known from ecological and conservation perspectives., (© 2022. Sociedade Entomológica do Brasil.)

Introgression is an important biological process affecting at least 10% of the extant species in the animal kingdom. Introgression significantly impacts inference of phylogenetic species relationships where a strictly binary tree model cannot adequately explain reticulate net-like species relationships. Here, we use phylogenomic approaches to understand patterns of introgression along the evolutionary history of a unique, nonmodel insect system: dragonflies and damselflies (Odonata). We demonstrate that introgression is a pervasive evolutionary force across various taxonomic levels within Odonata. In particular, we show that the morphologically "intermediate" species of Anisozygoptera (one of the three primary suborders within Odonata besides Zygoptera and Anisoptera), which retain phenotypic characteristics of the other two suborders, experienced high levels of introgression likely coming from zygopteran genomes. Additionally, we find evidence for multiple cases of deep inter-superfamilial ancestral introgression. [Gene flow; Odonata; phylogenomics; reticulate evolution.]., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Systematic Biologists.)

Background: The globe skimmer dragonfly (Pantala flavescens) is a notable Odonata insect distributed in nature fields and farmlands worldwide, and it is commonly recognized as a natural enemy because it preys on agricultural pests and health pests. As one of the sister groups of winged insects, odonatan species are key to understanding the evolution of insect wings., Findings: We present a high-quality reference genome of P. flavescens, which is the first chromosome-level genome in the Palaeoptera (Odonata and Ephemeroptera). The assembled genome size was 662 Mb, with a contig N50 of 16.2 Mb. Via Hi-C scaffolding, 648 Mb (97.9%) of contig sequences were clustered, ordered, and assembled into 12 large scaffolds, each corresponding to a natural chromosome. The X chromosome was identified by sequence coverage depth. The repetitive sequences and gene density of the X chromosome are similar to those of autosomal sequences, but the X chromosome shows a much lower degree of heterozygosity. Our analysis shows that the effective population size experienced 3 declining events, which may have been caused by climate change and environmental pollution., Conclusions: The genome of P. flavescens provides more information on the biology and evolution of insects and will help for the use of this species in pest control., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Insects comprise over half of the described species, and the acquisition of metamorphosis must have contributed to their diversity and prosperity. The order Odonata (dragonflies and damselflies) is among the most-ancestral insects with drastic morphological changes upon metamorphosis, in which understanding of the molecular mechanisms will provide insight into the evolution of incomplete and complete metamorphosis in insects. In order to identify metamorphosis-related genes in Odonata, we performed comprehensive RNA-sequencing of the blue-tailed damselfly Ischnura senegalensis at different developmental stages. Comparative RNA-sequencing analyses between nymphs and adults identified eight nymph-specific and seven adult-specific transcripts. RNA interference (RNAi) of these candidate genes demonstrated that three transcription factors, Krüppel homolog 1 ( Kr-h1 ), broad , and E93 play important roles in metamorphosis of both I. senegalensis and a phylogenetically distant dragonfly, Pseudothemis zonata E93 is essential for adult morphogenesis, and RNAi of Kr-h1 induced precocious metamorphosis in epidermis via up-regulation of E93 Precocious metamorphosis was also induced by RNAi of the juvenile hormone receptor Methoprene-tolerant ( Met ), confirming that the regulation of metamorphosis by the MEKRE93 (Met-Kr-h1-E93) pathway is conserved across diverse insects including the basal insect lineage Odonata. Notably, RNAi of broad produced unique grayish pigmentation on the nymphal abdominal epidermis. Survey of downstream genes for Kr-h1 , broad , and E93 uncovered that unlike other insects, broad regulates a substantial number of nymph-specific and adult-specific genes independently of Kr-h1 and E93 These findings highlight the importance of functional changes and rewiring of the transcription factors Kr-h1 , broad , and E93 in the evolution of insect metamorphosis., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Background: Evolutionary processes can cause strong spatial genetic signatures, such as local loss of genetic diversity, or conflicting histories from mitochondrial versus nuclear markers. Investigating these genetic patterns is important, as they may reveal obscured processes and players. The maternally inherited bacterium Wolbachia is among the most widespread symbionts in insects. Wolbachia typically spreads within host species by conferring direct fitness benefits, and/or by manipulating its host reproduction to favour infected over uninfected females. Under sufficient selective advantage, the mitochondrial haplotype associated with the favoured maternally-inherited symbiotic strains will spread (i.e. hitchhike), resulting in low mitochondrial genetic variation across the host species range., Method: The common bluetail damselfly (Ischnura elegans: van der Linden, 1820) has recently emerged as a model organism for genetics and genomic signatures of range expansion during climate change. Although there is accumulating data on the consequences of such expansion on the genetics of I. elegans, no study has screened for Wolbachia in the damselfly genus Ischnura. Here, we present the biogeographic variation in Wolbachia prevalence and penetrance across Europe and Japan (including samples from 17 populations), and from close relatives in the Mediterranean area (i.e. I. genei: Rambur, 1842; and I. saharensis: Aguesse, 1958)., Results: Our data reveal (a) multiple Wolbachia-strains, (b) potential transfer of the symbiont through hybridization, (c) higher infection rates at higher latitudes, and (d) reduced mitochondrial diversity in the north-west populations, indicative of hitchhiking associated with the selective sweep of the most common strain. We found low mitochondrial haplotype diversity in the Wolbachia-infected north-western European populations (Sweden, Scotland, the Netherlands, Belgium, France and Italy) of I. elegans, and, conversely, higher mitochondrial diversity in populations with low penetrance of Wolbachia (Ukraine, Greece, Montenegro and Cyprus). The timing of the selective sweep associated with infected lineages was estimated between 20,000 and 44,000 years before present, which is consistent with the end of the last glacial period about 20,000 years., Conclusions: Our findings provide an example of how endosymbiont infections can shape spatial variation in their host evolutionary genetics during postglacial expansion. These results also challenge population genetic studies that do not consider the prevalence of symbionts in many insects, which we show can impact geographic patterns of mitochondrial genetic diversity., (© 2021. The Author(s).)

Detection of environmental DNA (eDNA) has become a commonly used surveillance method for threatened or invasive vertebrates in both aquatic and terrestrial environments. However, most studies in this field favor vertebrate target species. Environmental DNA protocols can be especially useful for endangered invertebrates such as the Hine's emerald dragonfly (Somatochlora hineana) where conservation efforts have been greatly hindered by training, time, overall costs, and environmental impacts associated with conducting surveys in the calcareous fens occupied by this species. An essential step in developing such a protocol is to evaluate the dynamics of eDNA concentration under controlled conditions. We used the quantitative polymerase chain reaction (qPCR) to examine seasonal shifts in the persistence and net-accumulation of eDNA from captive S. hineana larvae in experimental mesocosms at temperatures corresponding with their overwintering (5.0 °C) and active (16.0 °C) seasons. Environmental DNA persisted longer at 5.0 °C but accumulated more readily at 16.0 °C. Differences in the accumulation and persistence of eDNA reflect differences in the longevity of eDNA at different temperatures and seasonal differences in larval S. hineana behavior. This study highlights the importance of considering how seasonal changes in temperature influence not only the speed of eDNA degradation but also the target species' eDNA shedding rates., (© 2021. The Author(s).)

Large-scale latitudinal studies that include both north and south edge populations and address sex differences are needed to understand how selection has shaped trait variation. We quantified the variation of flight-related morphological traits (body size, wing size, ratio between wing size and body size, and wing shape) along the whole latitudinal distribution of the damselfly Lestes sponsa, spanning over 2700 km. We tested predictions of geographic variation in the flight-related traits as a signature of: (1) stronger natural selection to improve dispersal in males and females at edge populations; (2) stronger sexual selection to improve reproduction (fecundity in females and sexual behaviors in males) at edge populations. We found that body size and wing size showed a U-shaped latitudinal pattern, while wing ratio showed the inverse shape. However, wing shape varied very little along the latitudinal gradient. We also detected sex-differences in the latitudinal patterns of variation. We discuss how latitudinal differences in natural and sexual selection regimes can lead to the observed quadratic patterns of variation in body and wing morphology via direct or indirect selection. We also discuss the lack of latitudinal variation in wing shape, possibly due to aerodynamic constraints., (© 2021. The Author(s).)

The order Odonata (dragonflies and damselflies) comprises diurnal insects with well-developed vision, showing diverse colors in adult wings and bodies. It is one of the most ancestral winged insect groups. Because Odonata species use visual cues to recognize each other, color patterns have been investigated from ecological and evolutionary viewpoints. Here we review the recent progress on molecular mechanisms of pigmentation, especially focused on light-blue coloration. Results from histology and pigment analysis showed that ommochrome pigments on the proximal layer and pteridine pigments on the distal layer of the epidermis are essential for light-blue coloration. We also summarize genes involved in the biosynthesis of three major insect pigments conserved across insects and discuss that gene-functional analysis deserves future studies., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dragonflies and damselflies are a charismatic, medium-sized insect order (~6300 species) with a unique potential to approach comparative research questions. Their taxonomy and many ecological traits for a large fraction of extant species are relatively well understood. However, until now, the lack of a large-scale phylogeny based on high throughput data with the potential to connect both perspectives has precluded comparative evolutionary questions for these insects. Here, we provide an ordinal hypothesis of classification based on anchored hybrid enrichment using a total of 136 species representing 46 of the 48 families or incertae sedis, and a total of 478 target loci. Our analyses recovered the monophyly for all three suborders: Anisoptera, Anisozygoptera and Zygoptera. Although the backbone of the topology was reinforced and showed the highest support values to date, our genomic data was unable to stronglyresolve portions of the topology. In addition, a quartet sampling approach highlights the potential evolutionary scenarios that may have shaped evolutionary phylogeny (e.g., incomplete lineage sorting and introgression) of this taxon. Finally, in light of our phylogenomic reconstruction and previous morphological and molecular information we proposed an updated odonate classification and define five new families (Amanipodagrionidae fam. nov., Mesagrionidae fam. nov., Mesopodagrionidae fam. nov., Priscagrionidae fam. nov., Protolestidae fam. nov.) and reinstate another two (Rhipidolestidae stat. res., Tatocnemididae stat. res.). Additionally, we feature the problematic taxonomic groupings for examination in future studies to improve our current phylogenetic hypothesis., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Colour polymorphisms are popular study systems among biologists interested in evolutionary dynamics, genomics, sexual selection and sexual conflict. In many damselfly groups, such as in the globally distributed genus Ischnura (forktails), sex-limited female colour polymorphisms occur in multiple species. Female-polymorphic species contain two or three female morphs, one of which phenotypically matches the male (androchrome or male mimic) and the other(s) which are phenotypically distinct from the male (heterochrome). These female colour polymorphisms are thought to be maintained by frequency-dependent sexual conflict, but their macroevolutionary histories are unknown, due to the lack of a robust molecular phylogeny. Here, we present the first time-calibrated phylogeny of Ischnura, using a multispecies coalescent approach (StarBEAST2) and incorporating both molecular and fossil data for 41 extant species (55% of the genus). We estimate the age of Ischnura to be between 13.8 and 23.4 millions of years, i.e. Miocene. We infer the ancestral state of this genus as female monomorphism with heterochrome females, with multiple gains and losses of female polymorphisms, evidence of trans-species female polymorphisms and a significant positive relationship between female polymorphism incidence and current geographic range size. Our study provides a robust phylogenetic framework for future research on the dynamic macroevolutionary history of this clade with its extraordinary diversity of sex-limited female polymorphisms., (Copyright © 2021. Published by Elsevier Inc.)

The evolution of sex chromosomes, and patterns of sex-biased gene expression and dosage compensation, are poorly known among early winged insects such as odonates. We assembled and annotated the genome of Ischnura elegans (blue-tailed damselfly), which, like other odonates, has a male-hemigametic sex-determining system (X0 males, XX females). By identifying X-linked genes in I. elegans and their orthologs in other insect genomes, we found homologies between the X chromosome in odonates and chromosomes of other orders, including the X chromosome in Coleoptera. Next, we showed balanced expression of X-linked genes between sexes in adult I. elegans, i.e. evidence of dosage compensation. Finally, among the genes in the sex-determining pathway only fruitless was found to be X-linked, while only doublesex showed sex-biased expression. This study reveals partly conserved sex chromosome synteny and independent evolution of dosage compensation among insect orders separated by several hundred million years of evolutionary history., (Copyright © 2021. Published by Elsevier Inc.)

AbstractAlthough natural selection often fluctuates across ontogeny, it remains unclear what conditions enable selection in one life-cycle stage to shape evolution in others. Organisms that undergo metamorphosis are useful for addressing this topic because their highly specialized life-cycle stages cannot always evolve independently despite their dramatic life-history transition. Using a comparative study of dragonflies, we examined three conditions that are hypothesized to allow selection in one stage to affect evolution in others. First, we tested whether lineages with less dramatic metamorphosis (e.g., hemimetabolous insects) lack the capacity for stage-specific evolution. Rejecting this hypothesis, we found that larval body shape evolves independently from selection on adult shape. Next, we evaluated whether stage-specific evolution is limited for homologous and/or coadapted structures. Indeed, we found that selection for larger wings is associated with the evolution of coadapted larval sheaths that store developing wing tissue. Finally, we assessed whether stage-specific evolution is restricted for traits linked to a single biochemical pathway. Supporting this hypothesis, we found that species with more wing melanization in the adult stage have evolved weaker melanin immune defenses in the larval stage. Thus, our results collectively show that natural selection in one stage imposes trait-dependent constraints on evolution in others.

Natural selection on juveniles is often invoked as a constraint on adult evolution, but it remains unclear when such restrictions will have their greatest impact. Selection on juveniles could, for example, mainly limit the evolution of adult traits that mostly develop prior to maturity. Alternatively, selection on juveniles might primarily constrain the evolution of adult traits that experience weak or context-dependent selection in the adult stage. Using a comparative study of dragonflies, I tested these hypotheses by examining how a species' larval habitat was related to the evolution of two adult traits that differ in development and exposure to selection: adult size and male ornamentation. Whereas adult size is fixed at metamorphosis and experiences consistent positive selection in the adult stage, ornaments develop throughout adulthood and provide context-dependent fitness benefits. My results show that species that develop in less stable larval habitats have smaller adult sizes and slower rates of adult size evolution. However, these risky larval habitats do not limit ornament expression or rates of ornament evolution. Selection on juveniles may therefore primarily affect the evolution of adult traits that mostly develop prior to maturity.

The order Odonata (dragonflies and damselflies) is among the most ancestral groups of winged insects with drastic morphological changes upon metamorphosis, and thus important for understanding evo-devo aspects of insects. However, basic developmental descriptions of Odonata have been scarce. In an attempt to establish the foundation of developmental and experimental biology of Odonata, we present an unprecedentedly comprehensive survey of dragonflies and damselflies, in total 158 larvae representing 49 species and 14 families, wherein morphological changes of all the final and/or penultimate instar larvae were photographed and monitored everyday. Although their morphology and development were diverse, we consistently identified two visually recognizable morphogenetic events in the final larval instar, namely start of wing expansion and onset of melanization on the wing sheaths, thereby categorizing the final instar into three stages. While the duration of the first stage ranged 4-66 days across diverse Odonata species, the second or third stages exhibited relatively small variation ranging 3-22 days or 1-8 days, respectively, probably reflecting the steady and irreversible metamorphosis process after stage 2. We also described other characteristic morphological changes during the larval development, although they were observed only in some Odonata species and lineages.

We present the results of the first-ever DNA barcoding study of odonates from the Maltese Islands. In total, 10 morphologically identified species were collected during a two-week long expedition in 2018. Eighty cytochrome c oxidase subunit I (COI) barcodes were obtained from the collected specimens. Intra- and interspecific distances ranged from 0.00% to 2.24% and 0.48% to 17.62%, respectively. Successful species identification based on ascribing a single morphological species to a single Barcode Index Number (BIN) was achieved for eight species (80%). In the case of two species, Ischnura genei and Anax parthenope , BINs were shared with other closely related species. The taxonomic status of I. genei is questionable and the phylogenetic relationship between A. imperator / parthenope is not clear. Further studies involving a series of adult specimens collected in a wide spatial range and nuclear markers are necessary to resolve these cases. Therefore, this dataset serves as an initial DNA barcode reference library for Maltese odonates, within a larger project: Aquatic Macroinvertebrates DNA Barcode Library of Malta.

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

Phenotypic plasticity can either hinder or promote adaptation to novel environments. Recent studies that have quantified alignments between plasticity, genetic variation, and divergence propose that such alignments may reflect constraints that bias future evolutionary trajectories. Here, we emphasize that such alignments may themselves be a result of natural selection and do not necessarily indicate constraints on adaptation. We estimated developmental plasticity and broad sense genetic covariance matrices (G) among damselfly populations situated along a latitudinal gradient in Europe. Damselflies were reared at photoperiod treatments that simulated the seasonal time constraints experienced at northern (strong constraints) and southern (relaxed constraints) latitudes. This allowed us to partition the effects of (1) latitude, (2) photoperiod, and (3) environmental novelty on G and its putative alignment with adaptive plasticity and divergence. Environmental novelty and latitude did not affect G, but photoperiod did. Photoperiod increased evolvability in the direction of observed adaptive divergence and developmental plasticity when G was assessed under strong seasonal time constraints at northern (relative to southern) photoperiod. Because selection and adaptation under time constraints is well understood in Lestes damselflies, our results suggest that natural selection can shape the alignment between divergence, plasticity, and evolvability., (© 2020 The Authors. Evolution published by Wiley Periodicals LLC on behalf of The Society for the Study of Evolution.)

Global warming is causing plastic and evolutionary changes in the phenotypes of ectotherms. Yet, we have limited knowledge on how the interplay between plasticity and evolution shapes thermal responses and underlying gene expression patterns. We assessed thermal reaction norm patterns across the transcriptome and identified associated molecular pathways in northern and southern populations of the damselfly Ischnura elegans. Larvae were reared in a common garden experiment at the mean summer water temperatures experienced at the northern (20°C) and southern (24°C) latitudes. This allowed a space-for-time substitution where the current gene expression levels at 24°C in southern larvae are a proxy for the expected responses of northern larvae under gradual thermal evolution to the predicted 4°C warming. Most differentially expressed genes showed fixed differences across temperatures between latitudes, suggesting that thermal genetic adaptation will mainly evolve through changes in constitutive gene expression. Northern populations also frequently showed plastic responses in gene expression to mild warming, while southern populations were much less responsive to temperature. Thermal responsive genes in northern populations showed to a large extent a pattern of genetic compensation, namely gene expression that was induced at 24°C in northern populations remained at a lower constant level in southern populations, and were associated with metabolic and translation pathways. There was instead little evidence for genetic assimilation of an initial plastic response to mild warming. Our data therefore suggest that genetic compensation rather than genetic assimilation may drive the evolution of plasticity in response to mild warming in this damselfly species., (© 2020 John Wiley & Sons Ltd.)

In community ecology, it is important to understand the distribution of communities along environmental and spatial gradients. However, it is common for the residuals of models investigating those relationships to be very high (> 50%). It is believed that species' intrinsic characteristics such as rarity can contribute to large residuals. The objective of this study is to test the relationship among communities and environmental and spatial predictors by evaluating the relative contribution of common and rare species to the explanatory power of models. Our hypothesis is that the residual of partition the variation of community matrix (varpart) models will decrease as rare species get removed. We used several environmental variables and spatial filters as varpart model predictors of fish and Zygoptera (Insecta: Odonata) communities in 109 and 141 Amazonian streams, respectively. We built a repetition structure, in which we gradually removed common and rare species independently. After the repetitions and removal of species, our hypothesis was not corroborated. In all scenarios, removing up to 50% of rare species did not reduce model residuals. Common species are important and rare species are irrelevant for understanding the relationships among communities and environmental and spatial gradients using varpart. Therefore, our findings suggest that studies using varpart with single sampling events that do not detect rare species can efficiently assess general distributional patterns of communities along environmental and spatial gradients. However, when the objectives concern conservation of biodiversity and functional diversity, rare species must be carefully assessed by other complementary methods, since they are not well represented in varpart models.

All natural populations show fluctuations in space or time. This is fundamental for the maintenance of biodiversity, as it allows species to coexist. Long-term ecological studies are rare, mainly due to logistics, but studies like the one presented below recognize the dimensionality of temporal change and the ecological processes that lead to shifts in community composition over time. Here, we used three sampling occasions from a dataset spanning 20 years where dragonflies in central Sweden were monitored. Our aim was to investigate how the prevalence of ecological and biological species traits varied over time measured as Community-level Weighted Means of trait values (CWM). Most CWM values varied significantly between years. Most of the traits changed between the second and the last sampling occasion, but not between the two first ones. These changes could be linked to major changes in species abundance. Our work indicates that fundamental shifts in community structure can occur over a short time, providing environmental drivers act on species turnover. In our case, Climate change and pH levels in lakes are most likely the most important factors.

The Synlestidae (Odonata: Zygoptera) of southern Africa comprise some highly localized species. All but one species are endemic to South Africa, and many to the Cape Floristic Region. Here we present the first phylogenetic reconstruction of the southern African Synlestidae using nuclear and mitochondrial molecular data. The genera Ecchlorolestes and Chlorolestes are monophyletic, and we propose that the Neotropical family Perilestidae consisting of two genera, Perilestes and Perissolestes, be sunk within Synlestidae. We discuss the intra-familial relationships for the southern African Synlestidae.

The larva of Agriocnemis minima Selys, 1879 is described and illustrated for the first time, based on reared specimens collected from Thailand. Selected larvae of Agrioncnemis Selys, 1877 were matched with their adults by DNA barcoding. The mitochondrial COI gene (658 bp) of three species (A. minima, A. femina femina (Brauer, 1868), and A. pygmaea (Rambur, 1842)) occurring in Thailand was analysed to confirm the species identification and to determine the association between the larva and adult stages. The larva of A. minima can be distinguished from known species by the following combination of characteristics: 1) long simple setae on the antennomeres I and II, 2) protrusion of the male cerci as long as 0.5× the S10, and 3) tufts of spiniform setae on the lateral occiput margin and on the ventral view of the compound eyes. Comparisons to known larvae of Agriocnemis and those of some other subfamily Agriocnemidinae members are also provided.

Phylogenetic relationships of basal insects remain a matter of discussion. In particular, the relationships among Ephemeroptera, Odonata and Neoptera are the focus of debate. In this study, we used a next-generation sequencing approach to reconstruct new mitochondrial genomes (mitogenomes) from 18 species of basal insects, including six representatives of Ephemeroptera and 11 of Odonata, plus one species belonging to Zygentoma. We then compared the structures of the newly sequenced mitogenomes. A tRNA gene cluster of IMQM was found in three ephemeropteran species, which may serve as a potential synapomorphy for the family Heptageniidae. Combined with published insect mitogenome sequences, we constructed a data matrix with all 37 mitochondrial genes of 85 taxa, which had a sampling concentrating on the palaeopteran lineages. Phylogenetic analyses were performed based on various data coding schemes, using maximum likelihood and Bayesian inferences under different models of sequence evolution. Our results generally recovered Zygentoma as a monophyletic group, which formed a sister group to Pterygota. This confirmed the relatively primitive position of Zygentoma to Ephemeroptera, Odonata and Neoptera. Analyses using site-heterogeneous CAT-GTR model strongly supported the Palaeoptera clade, with the monophyletic Ephemeroptera being sister to the monophyletic Odonata. In addition, a sister group relationship between Palaeoptera and Neoptera was supported by the current mitogenomic data.

Nowadays, stormwater sedimentation ponds are popular in stormwater management because of their ability to mitigate flooding and treat polluted runoff from e.g. roads. In addition, they may provide other ecosystem services such as biodiversity. These man-made habitats will inevitably be polluted and the organisms living therein may be negatively affected by the chemical cocktail present in both the water and sediment compartments. The present study explored DNA damage in dragonfly nymphs (Odonata, Anisoptera) living in highway sedimentation ponds in comparison with natural ponds. The concentrations of Polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs and metals were also determined in sediment samples from the different ponds. The results showed that DNA damage was significantly higher in dragonfly nymphs living in sedimentation ponds compared to nymphs living in natural ponds. DNA damage was also highly and significantly correlated with the pollution levels in the sediment, i.e., PAH and Zinc. Finally, we report the concentrations of various alkylated PAHs which appeared to be very dominant in the sedimentation ponds. Our results show that there may be a conflict between the sedimentation ponds' primary function of protecting natural water bodies from polluted runoff and their secondary function as habitats for organisms. Overall, we suggest that this must be considered when planning and designing stormwater measures.

The genus Franciscobasis Machado & Bedê, 2016 is endemic to the Serra da Canastra National Park in Minas Gerais state, Brazil. Two species of Franciscobasis were described simultaneously with the genus description: F. franciscoi and F. sonia, the latter described only from females. Through morphological and molecular analysis, we investigated if F. sonia may represent the young female of F. franciscoi. Resulting data did not present adequate differences between females to characterize them as different species. Therefore, we suggest that F. sonia is a junior synonym of F. franciscoi, and the female of F. franciscoi goes through a complex ontogenetic color change., Competing Interests: The authors have declared that no competing interests exist.

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 (Φ ST  > 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We have examined divergence times of the Antillean damselfly genus Hypolestes, to elucidate which mechanism of allopatric speciation, vicariance or long-distance dispersal, could better explain the currently observed disjunct distributions of this genus. Samples of the three extant species of the genus, Hypolestes clara (Jamaica), H. hatuey (Hispaniola) and H. trinitatis (Cuba), were collected. Mitochondrial and nuclear DNA gene fragments were amplified to reconstruct phylogenetic relationships and estimate divergence times in this genus. Hypolestes comprises currently three species, which consist in four geographically and genetically isolated lineages located in Jamaica, Hispaniola, Eastern Cuba and Central Cuba. Results of our analyses suggest that the three species diverged between ∼5.91 and 1.69 mya, and that the separation between the lineages from Central Cuba and Eastern Cuba occurred between ∼2.0 and 0.62 mya. Disjunct distributions in the genus Hypolestes can be better explained by a long-distance dispersal mechanism, since the divergence times of the three species do not coincide with the timeline formation of the geographic barriers between Cuba, Hispaniola and Jamaica. The Cuban lineages of H. trinitatis constitute different molecular operational taxonomic units (MOTU). The elevation of these MOTU to the species category requires the analysis of additional characters., (Copyright © 2019 Elsevier Inc. All rights reserved.)

To analyze genetic characters of migratory dragonflies, we used 10 microsatellite markers and a partial sequence of the mitochondrial gene Cytb to investigate genetic diversity and demographic history among 19 populations of P. flavescens in eastern the monsoon region of China. In a Bayesian clustering analysis of the microsatellite data, three distinct clades were present, and each population consisted of a mixture of individuals from the three clusters. An AMOVA of the data from both the microsatellite loci and Cytb revealed that genetic variation was mainly within each population. For the 543 individuals from the 19 regions, 77 unique haplotypes were obtained by DnaSP 4.0, and a median-joining network showed no obvious geographical pattern and displayed high gene flow and minimal population genetic structure among the 19 populations. According to a Mantel test, there was no significant association between genetic distance and geographic distribution and no isolation by distance. Mismatch distribution and neutrality tests showed no demographic expansion for the 19 populations. Microsatellite and mitochondrial DNA data suggested there was high gene flow and low differentiation among the populations. These results will help provide valuable information to study the migratory route of insects, especially important agricultural pests.

Although low levels of genetic structure are expected in highly widespread species, geographical and/or ecological factors can limit species distributions and promote population structure and morphological differentiation. In order to determine the effects of geographical isolation on population genetic structure and wing morphology, 281 individuals of the cosmopolitan odonate Pantala flavescens were collected from four continental (Central and South America) and five insular sites (Polynesian islands and the Maldives). COI sequences and eight microsatellite loci were used to characterize genetic diversity and genetic structure between and within locations. Linear and geometric morphometry were used to evaluate differences in the size and shape of wings. Genetic analysis showed a global genetic difference between the continental and insular sites. American locations did not show genetic structure, even in locations separated by a distance of 5000 km. Easter Island showed the lowest values of genetic diversity (mainly mitochondrial diversity) and the highest values of genetic differences compared to other insular and continental sites. Individuals from Easter Island showed smaller forewings, a different abdomen length to thorax length ratio, and a different configuration of anal loop in the hindwings. Thus, the greater isolation, smaller area, and young geological age seem to have determined the genetic and morphological differences in P. flavescens of Easter Island, where selection could promote a loss of migratory behavior and may improve other life history traits, such as reproduction. This work provides new insight into how microevolutionary processes operate in isolated populations of cosmopolitan species.

Matrona basilaris Selys, 1853 is a damselfly distributed mainly in mainland China. A total of 423 individuals from 48 populations covering almost the entire range were sampled to explore the genetic diversity, phylogeographic structure, and demographic dynamics of the species using sequences of three mitochondrial genes (COI, COII, and ND1) and a nuclear (ITS1 + 5.8 S + ITS2) gene. Phylogenetic tree, median-joining network, and BAPS analyses indicated a four-group division of the entire population, and the divergence event was estimated to have occurred in the middle Pleistocene. The diverse terrain of mainland China as well as past climatic oscillations were assumed to have shaped the current phylogeographic pattern of M. basilaris. Multiple lines of evidence supported population expansion in Group 1 and Group 2 but not in Group 3 or Group 4. The expansion times corresponded to the transition phase from the LIG (∼0.14-0.12 Mya) to the LGM (∼0.021-0.018 Mya). The pre-LGM expansion model reflected a different pattern affecting the historical dynamics of the population of East Asian species caused by Pleistocene climatic changes. Interestingly, Group 2 exhibited a disjunctive distribution pattern. The possible reasons were introgression caused by female-biased dispersal or human phoresy during construction of the Forbidden City during the Ming Dynasty of China.

The convergent evolution of analogous features is an evolutionary process occurring independently across the tree of life. From the evolution of echolocation, prehensile tail, viviparity, or winged flight, environmental factors often drive this astonishing phenomenon. However, convergent evolution is not always conspicuous or easily identified. Giant damselflies count among the largest flying insects on Earth, and have astonishing ecologies including orb-web spider plucking and oviposition in phytotelmata. One species occurs in the Afrotropics and 18 species are found in the Neotropics. Convergent evolution was historically hypothesized based on the ecological and morphological affinities of these two geographically distant lineages but was not supported by earlier phylogenetic inferences supporting their monophyly. Using a molecular supermatrix approach and a large selection of outgroups, we revisit and reject the monophyly of Afrotropical and Neotropical giant damselflies that is otherwise supported by a morphological phylogeny. Molecular divergence time estimation suggests an origin of Afrotropical giant damselflies in the late Paleogene, and of Neotropical ones at the Cretaceous/Paleogene boundary, thereby rejecting a long-standing West Gondwana vicariance hypothesis. The strong ecological and morphological resemblances between these two independent lineages represents an astonishing case of Amphi-Atlantic tropical convergent evolution., (© 2019 The Author(s). Evolution © 2019 The Society for the Study of Evolution.)

Damselflies and dragonflies, of the order Odonata, have distinct body plans and predatory abilities. Knowledge of their various evolutionary histories will allow for an understanding of the genetic and phenotypic evolution of insects. Mitogenomes are suitable materials to elucidate this, but the mitogenome of only a few odonates have been annotated. Herein, we report the complete mitogenome of nine odonates, including seven dragonflies and two damselflies, and a comprehensive analysis of the codon usage in 31 Odonata mitogenomes with the aim to estimate their evolutionary characteristics. Overall, a weak codon bias exists among odonate mitogenomes, although this favours AT-ending codons. Damselflies have a weaker codon usage bias than dragonflies, and 37 codons have significantly different usages. Both directional mutation and purifying selection shape damselfly and dragonfly mitogenomes. Although inevitable, directional mutation bias plays a minor role, whereas purifying selection pressure is the dominant evolutionary force. A higher selection pressure is observed in dragonflies than in damselflies, but it mainly acts on codon usage patterns rather than amino acid translation. Our findings suggest that dragonflies might have more efficient mitochondrial gene expression levels than damselflies, producing more proteins that support their locomotion and predatory abilities.

Many animals change their body color for visual signaling and environmental adaptation. Some dragonflies show wax-based color change and ultraviolet (UV) reflection, but the biochemical properties underlying the phenomena are totally unknown. Here we investigated the UV-reflective abdominal wax of dragonflies, thereby identifying very long-chain methyl ketones and aldehydes as unique and major wax components. Little wax was detected on young adults, but dense wax secretion was found mainly on the dorsal abdomen of mature males of Orthetrum albistylum and O. melania , and pruinose wax secretion was identified on the ventral abdomen of mature females of O. albistylum and Sympetrum darwinianum . Comparative transcriptomics demonstrated drastic upregulation of the ELOVL17 gene, a member of the fatty acid elongase gene family, whose expression reflected the distribution of very long-chain methyl ketones. Synthetic 2-pentacosanone, the major component of dragonfly's wax, spontaneously formed light-scattering scale-like fine structures with strong UV reflection, suggesting its potential utility for biomimetics., Competing Interests: RF, YY, MK, DI, RK, SY, TH An international patent on the synthesis method and application of very long chain methyl ketones and aldehydes was applied as PCT/JP2018/019559. NM, GO, YH, KY, TF No competing interests declared, (© 2019, Futahashi et al.)

Many Odonata species exhibit female-limited polymorphisms, where one morph is similar to the conspecific male in body color and other traits (andromorph), whereas one or more other morphs differ from the male (gynomorphs). Here we investigated the differentially expressed transcripts (DETs) among males and two female morph groups (gynomorphs and andromorphs) using RNA-seq to identify candidate transcripts encoding female-limited polymorphisms in the damselfly Ischnura senegalensis. Seven DETs that had significantly different expression levels between males and gynomorphs, but not between males and andromorphs, were identified. The expression levels of four of these candidate genes, doublesex (dsx), black, ebony, and chaoptin (chp), were selected for further analysis using qRT-PCR. Sequence analysis of the dsx amplicons revealed that this gene produced at least three transcripts. Two short transcripts were mainly expressed in males and andromorphs, whereas the long transcript was specifically expressed in both morph female groups; that is, the expression pattern of the dsx splice variants in andromorphs was an intermediate between that of males and gynomorphs. Because the dsx gene functions as a transcription factor that regulates the sex-specific expression of multiple genes, its splice variants in I. senegalensis may explain why the andromorph is female but exhibits some masculinized traits. Because we did not detect different coding sequences of the candidate genes among the different morphs, a diallelic genomic region controlling alternative splicing of dsx, thus determining female-limited polymorphism in I. senegalensis most likely lies in a non-coding region of the dsx gene or in a gene upstream of it.