Tropical Origin, Global Diversification, and Dispersal in the Pond Damselflies (Coenagrionoidea) Revealed by a New Molecular Phylogeny.

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.

Molecular mechanisms underlying metamorphosis in the most-ancestral winged insect.

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 zonataE93 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.

A molecular phylogeny of the Petaluridae (Odonata: Anisoptera): A 160-Million-Year-Old story of drift and extinction.

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.

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

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

Exploring chromosome evolution in 250 million year old groups of dragonflies and damselflies (Insecta:Odonata).

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.

A story of vicariance? how the geology of oceanic archipelagos influenced the evolutionary history of endemic damselflies.

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.

Mixed support for an alignment between phenotypic plasticity and genetic differentiation in damselfly wing shape.

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.

Deep Ancestral Introgression Shapes Evolutionary History of Dragonflies and Damselflies.

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.].

Reference genome for the American rubyspot damselfly, Hetaerina americana.

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

Genetic variation and population structure of a widely distributed damselfly (Ischnura senegalensis).

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.

Restricted X chromosome introgression and support for Haldane's rule in hybridizing damselflies.

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.

Genome assembly, sex-biased gene expression and dosage compensation in the damselfly Ischnura elegans.

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.

Natural Selection on Adults Has Trait-Dependent Consequences for Juvenile Evolution in Dragonflies.

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.

A molecularphylogeny offorktail damselflies(genus Ischnura)revealsa dynamic macroevolutionary history of female colour polymorphisms.

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.

Phylogeny and classification of Odonata using targeted genomics.

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.

First records raise questions: DNA barcoding of Odonata in the middle of the Mediterranean.

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.

Larval habitats impose trait-dependent limits on the direction and rate of adult evolution in dragonflies.

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.

Genetic compensation rather than genetic assimilation drives the evolution of plasticity in response to mild warming across latitudes in a damselfly.

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.

Mechanisms of allopatric speciation in an Antillean damselfly genus (Odonata, Zygoptera): Vicariance or long-distance dispersal?

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.

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

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