Reanalyzing the Palaeoptera problem – The origin of insect flight remains obscure

The phylogenetic relationships of the winged insect lineages – mayflies (Ephemeroptera), damselflies and dragonflies (Odonata), and all other winged insects (Neoptera) – are still controversial with three hypotheses supported by different datasets: Palaeoptera, Metapterygota and Chiastomyaria. Here, we reanalyze available phylogenomic data with a focus on detecting confounding and alternative signal. In this context, we provide a framework to quantitatively evaluate and assess incongruent molecular phylogenetic signal inherent in phylogenomic datasets. Despite overall support for the Palaeoptera hypothesis, we also found considerable signal for Chiastomyaria, which is not easily detectable by standardized tree inference approaches. Analyses of the accumulation of signal across gene partitions showed that signal accumulates gradually. However, even in case signal only slightly supported one over the other hypothesis, topologies inferred from large datasets switch from statistically strongly supported Palaeoptera to strongly supported Chiastomyaria. From a morphological point of view, Palaeoptera currently appears to be the best-supported hypothesis; however, recent analyses were restricted to head characters. Phylogenetic approaches covering all organ systems including analyses of potential functional or developmental convergence are still pending so that the Palaeoptera problem has to be considered an open question in insect systematics.     

Fossil evidence for key innovations in the evolution of insect diversity

Explaining the taxonomic richness of the insects, comprising over half of all described species, is a major challenge in evolutionary biology. Previously, several evolutionary novelties (key innovations) have been posited to contribute to that richness, including the insect bauplan, wings, wing folding and complete metamorphosis, but evidence over their relative importance and modes of action is sparse and equivocal. Here, a new dataset on the first and last occurrences of fossil hexapod (insects and close relatives) families is used to show that basal families of winged insects (Palaeoptera, e.g. dragonflies) show higher origination and extinction rates in the fossil record than basal wingless groups (Apterygota, e.g. silverfish). Origination and extinction rates were maintained at levels similar to Palaeoptera in the more derived Polyneoptera (e.g. cockroaches) and Paraneoptera (e.g. true bugs), but extinction rates subsequently reduced in the very rich group of insects with complete metamorphosis (Holometabola, e.g. beetles). Holometabola show evidence of a recent slow-down in their high net diversification rate, whereas other winged taxa continue to diversify at constant but low rates. These data suggest that wings and complete metamorphosis have had the most effect on family-level insect macroevolution, and point to specific mechanisms by which they have influenced insect diversity through time.     

Early axonogenesis in the embryo of a primitive insect,the silverfish Ctenolepisma longicaudata

The pattern of axon growth from the population of neurons that pioneers the major axon pathways in the central nervous system is highly conserved in winged insects. This study sought to determine whether the same pattern of axon growth is shared by an apterygotic insect, the silverfish. We have found that homologues to at least nine early differentiating winged insect neurons are present in the silverfish. The axon trajectories and the sequence of axon outgrowth from these neurons are very similar in silverfish and winged insects, suggesting that the pterygotic and apterygotic insects share a common developmental Bauplan for the construction of the central nervous system. Some of these neurons do show differences in several aspects of axon growth, including the relative timing of axonogenesis, the polarity of axon growth and the pattern of axon fasciculation. In addition, a major, early-appearing fascicle in the posterior commissure of the silverfish is pioneered by a neuron which does not appear to have an equivalent in the winged insects. These differences are similar in character to, albeit more pronounced than, differences previously reported between two winged insects, the fruitfly Drosophila and the grasshopper. Some of the features of early central axon growth, that set the silverfish embryo apart from the winged insects, are shared by crustacean embryos, providing support for the claim that insects and crustaceans share a common developmental Bauplan for the construction of central axonal pathways.     

The problem with "the Paleoptera Problem:" sense and sensitivity

While the monophyly of winged insects (Pterygota) is well supported, phylogenetic relationships among the most basal extant pterygote lineages are problematic. Ephemeroptera (mayflies) and Odonata (dragonflies) represent the two most basal extant lineages of winged insects, and determining their relationship with regard to Neoptera (remaining winged insects) is a critical step toward understanding insect diversification. A recent molecular analysis concluded that Paleoptera (Odonata Ephemeroptera) is monophyletic. However, we demonstrate that this result is supported only under a narrow range of alignment parameters. We have further tested the monophyly of Paleoptera using additional sequence data from 18SrDNA, 28S rDNA, and Histone 3 for a broader selection of taxa and a wider range of analytical methodologies. Our results suggest that the current suite of molecular data ambiguously resolve the three basal winged insect lineages and do not provide independent confirmation of Odonata + Neoptera as supported via morphological data.     

On the head morphology of Grylloblattodea (Insecta) and the systematic position of the order,with a new nomenclature for the head muscles of Dicondylia

External and internal head structures of adults of Galloisiana yuasai (Grylloblattodea) are described. The results are compared with conditions found in representatives of other lower neopteran lineages, notably in Austrophasma and Karoophasma (both Mantophasmatodea). Sutures and ridges of the head capsule are discussed. A new nomenclature for head muscles is presented for the entire Dicondylia (= Zygentoma + Pterygota). Galloisiana (like its sister taxon Grylloblatta) is mostly characterized by plesiomorphic features, such as the largely unspecialized orthopteroid mouthparts, the multisegmented filiform antennae, the presence of trabeculae tentorii, the absence of muscles associated with the antennal ampullae, the presence of musculus stipitalis transversalis (0mx11) and the presence of musculus tentoriofrontalis anterior (0te2). Autapomorphies of Grylloblattodea are: (i) compound eyes composed of only 60 ommatidia or less; (ii) a lacinia with a proximal tooth; (iii) a rounded submentum; (iv) loss of musculus craniohypopharyngealis (0hy3); and (v) loss of musculus labroepipharyngealis (0lb5). The phylogenetic evaluation of 104 characters of the head yields a branching pattern with Grylloblattodea as a sister group of Mantophasmatodea in clade Xenonomia. Putative synapomorphies of both taxa are: (i) a distinct angle (more than 60°) between the submentum and the mentum; (ii) posteriorly oriented labial palpi; (iii) a flat and lobe‐like hypopharynx with a suspensorium far ventrad of the anatomical mouth opening; (iv) loss of musculus tentorioparaglossalis (0la6); and (v) a connection between the antennal ampulla and the supraoesophageal ganglion containing nuclei. Xenonomia is placed in a clade with the two dictyopteran terminals. Another monophyletic group is Embioptera + Phasmatodea. Most branches of the single tree obtained in our analysis are weakly supported. The results clearly show that more data and a much broader taxon sampling are required to clarify the phylogenetic interrelationships of the lower neopteran orders. However, our results narrow down the spectrum of possible solutions, and represent a starting point for future phylogenetic analyses, with an extensive concatenated dataset.     

Topographic anatomy of ascending and descending neurons of the supraesophageal,meso- and metathoracic ganglia in paleo- and neopterous insects

Topographic anatomy of ascending (AN) and descending (DN) neurons of the supraesophageal and thoracic ganglia in the nervous system of winged insects (Pterygota), representatives of the infraclasses Palaeoptera (Odonata, Aeschna grandis, dragonfly) and Neoptera (Blattoptera, Periplaneta americana, cockroach), was studied. These insects differ in ecological niches, lifestyles, sets of behavioral complexes, levels of locomotor system development, evolutionary age and systematic position. Cell bodies and processes of ANs and DNs were stained with nickel chloride (NiCl₂), and their topography was studied on total preparations of the supraesophageal and thoracic ganglia. Unlike cockroaches, the dragonfly protocerebrum was found to contain DNs sending their processes to ocelli. Dragonfly DN processes exhibit a specific branching pattern in thoracic ganglia, with collaterals coming off both ipsi- and contralaterally. In cockroaches, collaterals of DN processes come off ipsilaterally. The AN cell bodies in dragonfly meso- and metathoracic ganglia lie both ipsi- and contralaterally relative to the ascending process, whereas in cockroaches most of the AN cell bodies in the same ganglia are located contralaterally. Substantial differences in the distrubution of DNs and ANs in insects with different manners of locomotion appear to reflect different degrees of control the supraesophageal ganglion exerts over the activity of segmental centers. This does not seem to be related to the evolutionary age of insects or their systematic position. Probably, different degrees of control over locomotion depend on the way of food acquisition: catching prey in the air in “paleopterous” dragonflies versus maneuverable walking or running over a solid substrate in “neopterous” cockroaches.     

Resolving insect phylogeny: The significance of cephalic structures of the Nannomecoptera in understanding endopterygote relationships

The Nannomecoptera are among the most enigmatic and controversial taxa of endopterygote insects, the phylogenetic resolution of which is crucial to understanding the evolution of neopteran insects. Once considered a subordinate lineage within the Mecoptera, renewed interest in nannochoristids has been prompted by evidence that the Nannomecoptera are not admissible to the clade of extant scorpion flies but are more likely to belong to the clade Siphonaptera + Nannomecoptera + Diptera. The overarching purpose of the present account is to provide novel and extensive morphological character traits in addition to those already existing for adult structures. The aim is to determine if these traits support molecular data sets that have been suggested elsewhere to clarify the phyletic position of Nannochoristidae. This account focuses on nannomecopteran larvae, which unlike those of other mecopterans have received little attention. Thus, the thrust of this investigation is to provide detailed anatomical data on nannochoristid larvae for a targeted inquiry into their phylogenetic affinities. The described characters are discussed and presented in a data matrix comprising representatives of all endopterygote orders. While the study is based primarily on the New Zealand species Nannochorista philpotti, it is proposed that all nannomecopteran larvae will prove to be similar to this taxon in most if not all structural features of significance to a higher-level phylogenetic context.     

Is Ellipura monophyletic? A combined analysis of basal hexapod relationships with emphasis on the origin of insects

Hexapoda includes 33 commonly recognized orders, most of them insects. Ongoing controversy concerns the grouping of Protura and Collembola as a taxon Ellipura, the monophyly of Diplura, a single or multiple origins of entognathy, and the monophyly or paraphyly of the silverfish (Lepidotrichidae and Zygentoma s.s.) with respect to other dicondylous insects. Here we analyze relationships among basal hexapod orders via a cladistic analysis of sequence data for five molecular markers and 189 morphological characters in a simultaneous analysis framework using myriapod and crustacean outgroups. Using a sensitivity analysis approach and testing for stability, the most congruent parameters resolve Tricholepidion as sister group to the remaining Dicondylia, whereas most suboptimal parameter sets group Tricholepidion with Zygentoma. Stable hypotheses include the monophyly of Diplura, and a sister group relationship between Diplura and Protura, contradicting the Ellipura hypothesis. Hexapod monophyly is contradicted by an alliance between Collembola, Crustacea and Ectognatha (i.e., exclusive of Diplura and Protura) in molecular and combined analyses.     

A biomechanical analysis of prognathous and orthognathous insect head capsules: evidence for a many‐to‐one mapping of form to function

Insect head shapes are remarkably variable, but the influences of these changes on biomechanical performance are unclear. Among ‘basal’ winged insects, such as dragonflies, mayflies, earwigs and stoneflies, some of the most prominent anatomical changes are the general mouthpart orientation, eye size and the connection of the endoskeleton to the head. Here, we assess these variations as well as differing ridge and sclerite configurations using modern engineering methods including multibody dynamics modelling and finite element analysis in order to quantify and compare the influence of anatomical changes on strain in particular head regions and the whole head. We show that a range of peculiar structures such as the genal/subgenal, epistomal and circumocular areas are consistently highly loaded in all species, despite drastically differing morphologies in species with forward‐projecting (prognathous) and downward‐projecting (orthognathous) mouthparts. Sensitivity analyses show that the presence of eyes has a negligible influence on head capsule strain if a circumocular ridge is present. In contrast, the connection of the dorsal endoskeletal arms to the head capsule especially affects overall head loading in species with downward‐projecting mouthparts. Analysis of the relative strains between species for each head region reveals that concerted changes in head substructures such as the subgenal area, the endoskeleton and the epistomal area lead to a consistent relative loading for the whole head capsule and vulnerable structures such as the eyes. It appears that biting‐chewing loads are managed by a system of strengthening ridges on the head capsule irrespective of the general mouthpart and head orientation. Concerted changes in ridge and endoskeleton configuration might allow for more radical anatomical changes such as the general mouthpart orientation, which could be an explanation for the variability of this trait among insects. In an evolutionary context, many‐to‐one mapping of strain patterns onto a relatively similar overall head loading indeed could have fostered the dynamic diversification processes seen in insects.     

New inferences from tree shape: numbers of missing taxa and population growth rates

The relative positions of branching events in a phylogeny contain information about evolutionary and population dynamic processes. We provide new summary statistics of branching event times and describe how these statistics can be used to infer rates of species diversification from interspecies trees or rates of population growth from intraspecies trees. We also introduce a phylogenetic method for estimating the level of taxon sampling in a clade. Different evolutionary models and different sampling regimes can produce similar patterns of branching events, so it is important to consider explicitly the model assumptions involved when making evolutionary inferences. Results of an analysis of the phylogeny of the mosquito-borne flaviviruses suggest that there could be several thousand currently unidentified viruses in this clade.     

The Palaeoptera Problem: Basal Pterygote Phylogeny Inferred from 18S and 28S rDNA Sequences

Monophyly of the pterygote insects is generally accepted, but the relationships among the three basal branches (Odonata, Ephemeroptera and Neoptera) remain controversial. The traditional view, to separate the pterygote insects in Palaeoptera (Odonata + Ephemeroptera) and Neoptera, based on the ability or inability to fold the wings over the abdomen, has been questioned. Various authors have used different sets of morphological characters in support of all three possible arrangements of the basal pterygote branches. We sequenced 18S and 28S rDNA from 18 species of Odonata, 8 species of Ephemeroptera, 2 species of Neoptera, and 1 species of Archaeognatha in our study. The new sequences, in combination with sequences from GenBank, have been used in a parsimony jackknife analysis resulting in strong support for a monophyletic Palaeoptera. Morphological evidence and the phylogenetic implications for understanding the origin of insect flight are discussed.     

Patterns of embryonic neurogenesis in a primitive wingless insect, the silverfish, Ctenolepisma longicaudata: comparison with those seen in flying insects

 Neurogenesis was examined in the central nervous system of embryos of the primitively wingless insect, the silverfish, Ctenolepisma longicaudata, using staining with toluidine blue (TB) and the incorporation of bromodeoxyuridine (BUdR). The silverfish has the same number and positioning of neuroblasts as seen in more advanced insects and the relative order in which the different neuroblasts segregate from the neuroectoderm is highly conserved between Ctenolepisma and the grasshopper, Schistocerca. Of the 31 different neuroblasts found in a thoracic segment, one (NB 6–3) has a much longer proliferative period in silverfish. Of the remainder, 14 have similar proliferative phases, while16 neuroblasts have extended their proliferative period by 10% of embryogenesis or greater in the grasshopper as compared with the silverfish. Both insects had similar periods of abdominal neurogenesis except that in the silverfish terminal ganglion a prominent set of neuroblasts continued dividing until close to hatching, possibly reflecting the importance of cercal sensory input in this insect. This comparison between silverfish and grasshopper shows that the shift from wingless to flying insects was not accompanied by the addition of any new neuronal lineages in the thorax. Instead, selected lineages underwent a proliferative expansion to supply the additional neurons presumably needed for flight. The expansion of specific thoracic lineages was accompanied by the reduction of the terminal abdominal lineages as flying insects began to de-emphasize their cercal sensory system. Received:16 March 1998 / Accepted: 21 May 1998     

Biochemical and molecular biological aspects of silverfish allergens

Insects and insect-derived materials have been implicated as a risk factor for sensitization and subsequent elicitation of allergic rhinitis and allergic bronchial asthma. During the last decades, insects other than those known as allergenic, were investigated for their potential role in inducing and triggering an IgE immune response. Among these, the silverfish, an insect belonging to the Thysanura order, appeared to be of particular interest. Silverfish (Lepisma saccharina) is the most primitive living insect, and represents a descendent of the ancestral wingless insects. They are 3-12 mm long, have three tail feelers and are covered with shiny scales. They shun light and need a humid environment and their diet consists of carbohydrate materials such as paper and book-binding glue, crumbs of bread and flour. Because of these features, silverfish finds an optimal habitat both in dwellings and workplaces and in spite of its antiquity, silverfish has succeeded in exploiting the new opportunity created by man. Although its importance significantly increased when it has been demonstrated that house dust contains significant silverfish levels even in houses where the inhabitants were unaware of its presence, no silverfish extract for diagnosis of allergic diseases is commercially available yet. Identification of optimal extraction conditions and characterization of allergenic extracts are the first steps to obtain an effective allergen preparation suitable for diagnosis and therapy, and will be useful as a reference preparation for assessing silverfish exposure in different indoor environments. It has been cloned and characterized a silverfish tropomyosin, named Lep s 1, which represents the first allergen identified in silverfish extract and can be regarded as a molecule cross-reactive among inhalant and edible invertebrates allergenic sources. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.     

Eye movements and target fixation during dragonfly prey-interception flights

The capture of flying insects by foraging dragonflies is a highly accurate, visually guided behavior. Rather than simply aiming at the prey’s position, the dragonfly aims at a point in front of the prey, so that the prey is intercepted with a relatively straight flight trajectory. To better understand the neural mechanisms underlying this behavior, we used high-speed video to quantify the head and body orientation of dragonflies (female Erythemis simplicicollis flying in an outdoor flight cage) relative to an artificial prey object before and during pursuit. The results of our frame-by-frame analysis showed that during prey pursuit, the dragonfly adjusts its head orientation to maintain the image of the prey centered on the “crosshairs” formed by the visual midline and the dorsal fovea, a high acuity streak that crosses midline at right angles about 60° above the horizon. The visual response latencies to drifting of the prey image are remarkably short, ca. 25 ms for the head and 30 ms for the wing responses. Our results imply that the control of the prey-interception flight must include a neural pathway that takes head position into account.     

Molecular systematics and morphological character evolution of the Condamineeae (Rubiaceae)

? Premise of the study: The Condamineeae have in previous molecular studies been shown to be part of an early-divergent clade within the subfamily Ixoroideae, together with the tribes Calycophylleae, and Hippotideae, and genera of the former Cinchoneae and Rondeletieae. Generic relationships within this clade have, however, remained largely unresolved. ? Methods: In this study, the systematics of the Condamineeae was further examined by phylogenetic reconstruction of six cpDNA regions and one nrDNA region using parsimony and Bayesian Markov chain Monte Carlo inference. Morphological character evolution within the tribe was assessed by ancestral state reconstruction using likelihood optimization of characters onto Bayesian trees. ? Key results: Calycophylleae appears polyphyletic. "Hippotideae" is monophyletic but nested within the Condamineeae. The phylogenetic hypotheses presented support a resurrection of the genera Holtonia, Schizocalyx, and Semaphyllanthe. Furthermore, Bathysa is found to be polyphyletic, Tresanthera is found nested within Rustia, and the taxonomically disputed genus Dialypetalanthus is here shown to be sister to a Bothriospora-Wittmackanthus clade. Morphological ancestral state reconstructions indicate that protogyny have evolved at least two times within the tribe and that indehiscent fruits, loculicidal fruit dehiscence, and intrapetiolar stipules have evolved independently several times. The occurrence of calycophylls (leaf-like calyx lobes), poricidal anthers, and winged seeds also appear homoplastic within the tribe. ? Conclusions: A diagnosis and delimitation of the tribe Condamineeae is presented, with taxonomic proposals to synonymize Tresanthera and to transfer several species of Bathysa as well as Phitopis to a resurrected Schizocalyx.     

The phylogenetic relevance of thoracic musculature: a case study including a description of the thorax anatomy of Zygoptera (Insecta: Odonata) larvae

New morphological techniques allow for the evaluation of novel character systems that are potentially important for phylogenetic analysis. Only a few studies so far have used character systems from the insect thorax for phylogenetics; the reasons for this might include a lack of common terminology or established homology for pterygote insect thorax musculature. Still, recent studies have proposed common terminology and hypotheses of homology, now allowing for an evaluation of thoracic morphological character systems among the groups of winged insects. Using X ‐ray microtomography (μCT) we present a detailed study of the thorax musculature of O donata as an important phylogenetic character system, with a matrix of 298 characters with 697 character states, including novel data from the thoracic anatomy of eight damselfly larvae. We also included additional O donata, E phemeroptera and N eoptera taxa from the literature and demonstrate the phylogenetic relevance of this character system by reproducing phylogenetic topologies of established relationships. We also compared high‐resolution data from O donata larvae from our study and from recent literature with data from older literature in the adult O donata. All major clades were successfully recovered, (e.g. O donata, E piprocta, A nisoptera and Z ygoptera) with high node support, but obtained higher phylogenetic resolution with the larval data. The best phylogenetic resolution was achieved by combining the adult and larval characters. The taxon sampling and character matrix is the largest to date and underlines the potential relevance of the thorax musculature as an important phylogenetic character system.     

CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution

Despite previous developmental studies on basally branching wingless insects and crustaceans, the evolutionary origin of insect wings remains controversial. Knowledge regarding genetic regulation of tissues hypothesized to have given rise to wings would help to elucidate how ancestral development changed to allow the evolution of true wings. However, genetic tools available for basally branching wingless species are limited. The firebrat Thermobia domestica is an apterygote species, phylogenetically related to winged insects. T. domestica presents a suitable morphology to investigate the origin of wings, as it forms the tergal paranotum, from which wings are hypothesized to have originated. Here we report the first successful CRISPR/Cas9-based germline genome editing in T. domestica. We provide a technological platform to understand the development of tissues hypothesized to have given rise to wings in an insect with a pre-wing evolution body plan.     

A dual layer hair array of the brown lacewing: repelling water at different length scales

Additional weight due to contamination (water and/or contaminating particles) can potentially have a detrimental effect on the flight capabilities of large winged insects such as butterflies and dragonflies. Insects where the wing surface area-body mass ratio is very high will be even more susceptible to these effects. Water droplets tend to move spontaneously off the wing surface of these insects. In the case of the brown lacewing, the drops effectively encounter a dual bed of hair springs with a topographical structure which aids in the hairs resisting penetration into water bodies. In this article, we demonstrate experimentally how this protective defense system employed by the brown lacewing (Micromus tasmaniae) aids in resisting contamination from water and how the micro- and nanostructures found on these hairs are responsible for quickly shedding water from the wing which demonstrates an active liquid-repelling surface.     

Development of embryonic membranes in the silverfish Lepisma saccharina linnaeus (insecta: Zygentoma, Lepismatidae)

Development and fate of embryonic membranes in the silverfish Lepisma saccharina was examined throughout embryogenesis. The amnioserosal folds first arise as serosal folds that are completed by the later addition of the amnion from the embryo's margins as in archaeognaths. The close link between production of the amnion and formation of the folds should not be assigned to Dicondylia but to Pterygota as an autapomorphy. During fold formation, folding of embryonic membranes beneath the embryo is less extensive and the ventral cupping of the embryo plays a larger role comparable to that occurring in archaeognath embryos. In L. saccharina, the embryonic membrane pore (the amniopore) varies in its manner of closure, either by complete fusion of serosal folds or by formation of a serosal cuticular plug between them as in archaeognaths. Although, in many aspects of its embryogenesis, L. saccharina retains the primitiveness of archaeognaths, its amnioserosal folds persist and are well integrated into its embryogenesis as the amnioserosal fold-amniotic cavity system is established and as occurs in many pterygote embryos; this may be thus regarded as an autapomorphy of Dicondylia.