Insect antennae: Coupling blood pressure with cuticle deformation to control movement.

Insect antennae are hollow, blood-filled fibers with complex shape. Muscles in the two basal segments control antennal movement, but the rest (flagellum) is muscle-free. The insect can controllably flex, twist, and maneuver its antennae laterally. To explain this behavior, we performed a comparative study of structural and tensile properties of the antennae of Periplaneta americana (American cockroach), Manduca sexta (Carolina hawkmoth), and Vanessa cardui (painted lady butterfly). These antennae demonstrate a range of distinguishable tensile properties, responding either as brittle or strain-adaptive fibers that stiffen when stretched. Scanning electron microscopy and high-speed imaging of antennal breakup during stretching revealed complex coupling of blood pressure and cuticle deformation in antennae. A generalized Lamé theory of solid mechanics was developed to include the force-driven deformation of blood-filled antennal tubes. We validated the theory against experiments with artificial antennae with no adjustable parameters. Blood pressure increased when the insect inflated its antennae or decreased below ambient pressure when an external tensile load was applied to the antenna. The pressure-cuticle coupling can be controlled through changes of the blood volume in the antennal lumen. In insects that do not fill the antennal lumen with blood, this blood pressure control is lacking, and the antennae react only by muscular activation. We suggest that the principles we have discovered for insect antennae apply to other appendages that share a leg-derived ancestry. Our work offers promising new applications for multifunctional fiber-based microfluidics that could transport fluids and be manipulated by the same fluid on demand. STATEMENT OF SIGNIFICANCE: Insect antennae are blood-filled, segmented fibers with muscles in the two basal segments. The long terminal segment is muscle-free but can be flexed. To explain this behavior, we examined structure-function relationships of antennae of cockroaches, hawkmoths, and butterflies. Hawkmoth antennae behaved as brittle fibers, but butterfly and cockroach antennae showed strain-adaptive behavior like fibers that stiffen when stretched. Videomicroscopy of antennal breakup during stretching revealed complex coupling of blood pressure and cuticle deformation. Our solid mechanics model explains this behavior. Because antennae are leg-derived appendages, we suggest that the principles we found apply to other appendages of leg-derived ancestry. Our work offers new applications for multifunctional fiber-based microfluidics that could transport fluids and be manipulated by the fluid on demand.

Structural organization of xanthine crystals in the median ocellus of a member of the ancestral insect group Archaeognatha.

Biogenic purine crystals function in vision as mirrors, multilayer reflectors and light scatterers. We investigated a light sensory organ in a primarily wingless insect, the jumping bristletail Lepismachilis rozsypali (Archaeognatha), an ancestral group. The visual system of this animal comprises two compound eyes, two lateral ocelli, and a median ocellus, which is located on the front of the head, pointing downwards to the ground surface. We determined that the median ocellus contains crystals of xanthine, and we obtained insights into their function. To date, xanthine biocrystals have only been found in the Archaeognatha. We performed a structural analysis, using reflection light microscopy, cryo-FIB-SEM, microCT and cryo-SEM. The xanthine crystals cover the bottom of a bowl-shaped volume in the median ocellus, in analogy to a tapetum, and reflect photons to light-sensitive receptors that are spread in the volume without apparent order or preferential orientation. We infer that the median ocellus operates as an irregular multifocal reflector, which is not capable of forming images. A possible function of this organ is to improve photon capture, and by so doing assess distances from the ground surface when jumping by determining changes in the intensity and contrast of the incident light.

Wing hearts in four-winged Ultrabithorax-mutant flies-the role of Hox genes in wing heart specification.

Wings are probably the most advanced evolutionary novelty in insects. In the fruit fly Drosophila melanogaster, proper development of wings requires the activity of so-called wing hearts located in the scutellum of the thorax. Immediately after the imaginal ecdysis, these accessory circulatory organs remove hemolymph and apoptotic epidermal cells from the premature wings through their pumping action. This clearing process is essential for the formation of functional wing blades. Mutant flies that lack intact wing hearts are flightless and display malformed wings. The embryonic wing heart progenitors originate from two adjacent parasegments corresponding to the later second and third thoracic segments. However, adult dipterian flies harbor only one pair of wings and only one pair of associated wing hearts in the second thoracic segment. Here we show that the specification of WHPs depends on the regulatory activity of the Hox gene Ultrabithorax. Furthermore, we analyzed the development of wing hearts in the famous four-winged Ultrabithorax (Ubx) mutant, which was first discovered by Ed Lewis in the 1970s. In these flies, the third thoracic segment is homeotically transformed into a second thoracic segment resulting in a second pair of wings instead of the club-shaped halteres. We show that a second pair of functional wing hearts is formed in the transformed third thoracic segment and that all wing hearts originate from the wild-type population of wing heart progenitor cells.

Four myriapod relatives - but who are sisters? No end to debates on relationships among the four major myriapod subgroups.

BACKGROUND: Phylogenetic relationships among the myriapod subgroups Chilopoda, Diplopoda, Symphyla and Pauropoda are still not robustly resolved. The first phylogenomic study covering all subgroups resolved phylogenetic relationships congruently to morphological evidence but is in conflict with most previously published phylogenetic trees based on diverse molecular data. Outgroup choice and long-branch attraction effects were stated as possible explanations for these incongruencies. In this study, we addressed these issues by extending the myriapod and outgroup taxon sampling using transcriptome data. RESULTS: We generated new transcriptome data of 42 panarthropod species, including all four myriapod subgroups and additional outgroup taxa. Our taxon sampling was complemented by published transcriptome and genome data resulting in a supermatrix covering 59 species. We compiled two data sets, the first with a full coverage of genes per species (292 single-copy protein-coding genes), the second with a less stringent coverage (988 genes). We inferred phylogenetic relationships among myriapods using different data types, tree inference, and quartet computation approaches. Our results unambiguously support monophyletic Mandibulata and Myriapoda. Our analyses clearly showed that there is strong signal for a single unrooted topology, but a sensitivity of the position of the internal root on the choice of outgroups. However, we observe strong evidence for a clade Pauropoda+Symphyla, as well as for a clade Chilopoda+Diplopoda. CONCLUSIONS: Our best quartet topology is incongruent with current morphological phylogenies which were supported in another phylogenomic study. AU tests and quartet mapping reject the quartet topology congruent to trees inferred with morphological characters. Moreover, quartet mapping shows that confounding signal present in the data set is sufficient to explain the weak signal for the quartet topology derived from morphological characters. Although outgroup choice affects results, our study could narrow possible trees to derivatives of a single quartet topology. For highly disputed relationships, we propose to apply a series of tests (AU and quartet mapping), since results of such tests allow to narrow down possible relationships and to rule out confounding signal.

The Insect Circulatory System: Structure, Function, and Evolution.

Although the insect circulatory system is involved in a multitude of vital physiological processes, it has gone grossly understudied. This review highlights this critical physiological system by detailing the structure and function of the circulatory organs, including the dorsal heart and the accessory pulsatile organs that supply hemolymph to the appendages. It also emphasizes how the circulatory system develops and ages and how, by means of reflex bleeding and functional integration with the immune system, it supports mechanisms for defense against predators and microbial invaders, respectively. Beyond that, this review details evolutionary trends and novelties associated with this system, as well as the ways in which this system also plays critical roles in thermoregulation and tracheal ventilation in high-performance fliers. Finally, this review highlights how novel discoveries could be harnessed for the control of vector-borne diseases and for translational medicine, and it details principal knowledge gaps that necessitate further investigation.

Hypothesis on monochromatic vision in scorpionflies questioned by new transcriptomic data.

In the scorpionfly Panorpa, a recent study suggested monochromatic vision due to evidence of only a single opsin found in transcriptome data. To reconsider this hypothesis, the present study investigates opsin expression using transcriptome data of 21 species including representatives of all major lineages of scorpionflies (Mecoptera) and of three families of their closest relatives, the fleas (Siphonaptera). In most mecopteran species investigated, transcripts encode two opsins with predicted peak absorbances in the green, two in the blue, and one in the ultraviolet spectral region. Only in groups with reduced or absent ocelli, like Caurinus and Apteropanorpa, less than four visual opsin messenger RNAs have been identified. In addition, we found a Rh7-like opsin in transcriptome data derived from larvae of the mecopteran Nannochorista, and in two flea species. Peropsin expression was observed in two mecopterans. In light of these new data, we question the hypothesis on monochromatic vision in the genus Panorpa. In a broader phylogenetic perspective, it is suggested that the common ancestor of the monophyletic taxon Antliophora (Diptera, Mecoptera and Siphonaptera) possessed the full set of visual opsins, a Rh7-like opsin, and in addition a pteropsin as well as a peropsin. In the course of evolution individual opsins were likely lost in several lineages of this clade.

Beyond aerodynamics: The critical roles of the circulatory and tracheal systems in maintaining insect wing functionality.

Insect wings consist almost entirely of lifeless cuticle; yet their veins host a complex multimodal sensory apparatus and other tissues that require a continuous supply of water, nutrients and oxygen. This review provides a survey of the various living components in insect wings, as well as the specific contribution of the circulatory and tracheal systems to provide all essential substances. In most insects, hemolymph circulates through the veinal network in a loop flow caused by the contraction of accessory pulsatile organs in the thorax. In other insects, hemolymph oscillates into and out of the wings due to the complex interaction of several factors, such as heartbeat reversal, intermittent pumping of the accessory pulsatile organs in the thorax, and the elasticity of the wall of a special type of tracheae. A practically unexplored subject is the need for continuous hydration of the wing cuticle to retain its flexibility and toughness, including the associated problem of water loss due to evaporation. Also, widely neglected is the influence of the hemolymph mass and the circulating flow in the veins on the aerodynamic properties of insect wings during flight. Ventilation of the extraordinarily long wing tracheae is probably accomplished by intricate interactions with the circulatory system, and by the exchange of oxygen via cutaneous respiration.

Octopaminergic innervation and a neurohaemal release site in the antennal heart of the locust Schistocerca gregaria.

A detailed account is given by the octopaminergic innervation of the antennal heart in Schistocerca gregaria using various immunohistochemical methods. Anterograde axonal filling illustrates the unilateral innervation on the medial ventral surface of the pumping muscle of the antennal heart via the paired corpora cardiaca nerve III. In addition, antibody staining revealed that ascending axons of this nerve terminate at the ampullae of the antennal heart forming synaptoid structures and extensive neurohaemal release sites. Due to the innervation by two dorsal unpaired median neurons, the presence of the biogenic amines octopamine and tyramine could be visualized by immunocytochemistry in an insect antennal heart for the first time. The data suggest that tyramine acts as a precursor and not purely as an independent transmitter. While the octopaminergic fibers innervating the pumping muscle of the antennal heart indicate a cardioregulatory role, we conclude that octopamine released from the neurohaemal area is pumped into the antennae and an involvement in the modulation of the antennal sensory sensitivity is discussed.

The ocelli of Archaeognatha (Hexapoda): functional morphology, pigment migration and chemical nature of the reflective tapetum.

The ocelli of Archaeognatha, or jumping bristletails, differ from typical insect ocelli in shape and field of view. Although the shape of the lateral ocelli is highly variable among species, most Machiloidea have sole-shaped lateral ocelli beneath the compound eyes and a median ocellus that is oriented downward. This study investigated morphological and physiological aspects of the ocelli of Machilis hrabei and Lepismachilis spp. The light-reflecting ocellar tapetum in M. hrabei is made up of xanthine nanocrystals, as demonstrated by confocal Raman spectroscopy. Pigment granules in the photoreceptor cells move behind the tapetum in the dark-adapted state. Such a vertical pigment migration in combination with a tapetum has not been described for any insect ocellus so far. The pigment migration has a dynamic range of approximately 4 log units and is maximally sensitive to green light. Adaptation from darkness to bright light lasts over an hour, which is slow compared with the radial pupil mechanism in some dragonflies and locusts.

Morphological and Genetic Analysis of the Acerentomon doderoi Group (Protura: Acerentomidae) with Description of A. christiani sp. nov.

Acerentomon christiani sp. nov. is described from Vienna, Austria. The new species is a member of the "doderoi" group, characterized by the presence of seta x on tergite VII. It is most similar to A. gallicum, A. brevisetosum and A. tenuisetosum, but differs from these species in the length of foretarsal sensillum c and certain other chaetotaxic measurements and indices. In addition to the morphological description, the DNA barcoding region of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) and the 28S ribosomal RNA of the new species are provided. The morphological characters and the barcode of the new species are discussed in comparison to those of other Acerentomon species. An identification key to all known Acerentomon spp. of the "doderoi" group is given.

A new kind of auxiliary heart in insects: functional morphology and neuronal control of the accessory pulsatile organs of the cricket ovipositor.

INTRODUCTION: In insects, the pumping of the dorsal heart causes circulation of hemolymph throughout the central body cavity, but not within the interior of long body appendages. Hemolymph exchange in these dead-end structures is accomplished by special flow-guiding structures and/or autonomous pulsatile organs ("auxiliary hearts"). In this paper accessory pulsatile organs for an insect ovipositor are described for the first time. We studied these organs in females of the cricket Acheta domesticus by analyzing their functional morphology, neuroanatomy and physiological control. RESULTS: The lumen of the four long ovipositor valves is subdivided by longitudinal septa of connective tissue into efferent and afferent hemolymph sinuses which are confluent distally. The countercurrent flow in these sinuses is effected by pulsatile organs which are located at the bases of the ovipositor valves. Each of the four organs consists of a pumping chamber which is compressed by rhythmically contracting muscles. The morphology of the paired organs is laterally mirrored, and there are differences in some details between the dorsal and ventral organs. The compression of the pumping chambers of each valve pair occurs with a left-right alternating rhythm with a frequency of 0.2 to 0.5 Hz and is synchronized between the dorsal and ventral organs. The more anteriorly located genital chamber shows rhythmical lateral movements simultaneous to those of the ovipositor pulsatile organs and probably supports the hemolymph exchange in the abdominal apex region. The left-right alternating rhythm is produced by a central pattern generator located in the terminal ganglion. It requires no sensory feedback for its output since it persists in the completely isolated ganglion. Rhythm-modulating and rhythm-resetting interneurons are identified in the terminal ganglion. CONCLUSION: The circulatory organs of the cricket ovipositor have a unique functional morphology. The pumping apparatus at the base of each ovipositor valve operates like a bellow. It forces hemolymph via sinuses delimited by thin septa of connective tissue in a countercurrent flow through the valve lumen. The pumping activity is based on neurogenic control by a central pattern generator in the terminal ganglion.

Where taxonomy based on subtle morphological differences is perfectly mirrored by huge genetic distances: DNA barcoding in Protura (Hexapoda).

BACKGROUND: Protura is a group of tiny, primarily wingless hexapods living in soil habitats. Presently about 800 valid species are known. Diagnostic characters are very inconspicuous and difficult to recognize. Therefore taxonomic work constitutes an extraordinary challenge which requires special skills and experience. Aim of the present pilot project was to examine if DNA barcoding can be a useful additional approach for delimiting and determining proturan species. METHODOLOGY AND PRINCIPAL FINDINGS: The study was performed on 103 proturan specimens, collected primarily in Austria, with additional samples from China and Japan. The animals were examined with two markers, the DNA barcoding region of the mitochondrial COI gene and a fragment of the nuclear 28S rDNA (Divergent Domain 2 and 3). Due to the minuteness of Protura a modified non-destructive DNA-extraction method was used which enables subsequent species determination. Both markers separated the examined proturans into highly congruent well supported clusters. Species determination was performed without knowledge of the results of the molecular analyses. The investigated specimens comprise a total of 16 species belonging to 8 genera. Remarkably, morphological determination in all species exactly mirrors molecular clusters. The investigation revealed unusually huge genetic COI distances among the investigated proturans, both maximal intraspecific distances (0-21.3%), as well as maximal congeneric interspecifical distances (up to 44.7%). CONCLUSIONS: The study clearly demonstrates that the tricky morphological taxonomy in Protura has a solid biological background and that accurate species delimitation is possible using both markers, COI and 28S rDNA. The fact that both molecular and morphological analyses can be performed on the same individual will be of great importance for the description of new species and offers a valuable new tool for biological and ecological studies, in which proturans have generally remained undetermined at species level.

Decisive data sets in phylogenomics: lessons from studies on the phylogenetic relationships of primarily wingless insects.

Phylogenetic relationships of the primarily wingless insects are still considered unresolved. Even the most comprehensive phylogenomic studies that addressed this question did not yield congruent results. To get a grip on these problems, we here analyzed the sources of incongruence in these phylogenomic studies by using an extended transcriptome data set. Our analyses showed that unevenly distributed missing data can be severely misleading by inflating node support despite the absence of phylogenetic signal. In consequence, only decisive data sets should be used which exclusively comprise data blocks containing all taxa whose relationships are addressed. Additionally, we used Four-cluster Likelihood Mapping (FcLM) to measure the degree of congruence among genes of a data set, as a measure of support alternative to bootstrap. FcLM showed incongruent signal among genes, which in our case is correlated neither with functional class assignment of these genes nor with model misspecification due to unpartitioned analyses. The herein analyzed data set is the currently largest data set covering primarily wingless insects, but failed to elucidate their interordinal phylogenetic relationships. Although this is unsatisfying from a phylogenetic perspective, we try to show that the analyses of structure and signal within phylogenomic data can protect us from biased phylogenetic inferences due to analytical artifacts.

The bHLH transcription factor hand is required for proper wing heart formation in Drosophila.

The Hand basic helix-loop-helix transcription factors play an important role in the specification and patterning of various tissues in vertebrates and invertebrates. Here, we have investigated the function of Hand in the development of the Drosophila wing hearts which consist of somatic muscle cells as well as a mesodermally derived epithelium. We found that Hand is essential in both tissues for proper organ formation. Loss of Hand leads to a reduced number of cells in the mature organ and loss of wing heart functionality. In wing heart muscles Hand is required for the correct positioning of attachment sites, the parallel alignment of muscle cells, and the proper orientation of myofibrils. At the protein level, α-Spectrin and Dystroglycan are misdistributed suggesting a defect in the costameric network. Hand is also required for proper differentiation of the wing heart epithelium. Additionally, the handC-GFP reporter line is not active in the mutant suggesting an autoregulatory role of Hand in wing hearts. Finally, in a candidate-based RNAi mediated knock-down approach we identified Daughterless and Nautilus as potential dimerization partners of Hand in wing hearts.

In vivo imaging of Drosophila wing heart development during pupal stages.

Wing hearts are small pumping organs that maintain the flow of hemolymph through the wing veins of insects. In Drosophila, these organs consist of parallel oriented muscle cells and a simple epithelium of connective tissue. Both tissues originate from eight embryonic wing heart progenitors (WHPs), which remain dormant until late larval stages. Most of the differentiation and maturation takes place during the pupal stage following head eversion. In this study, we have used the tissue specific expression of Gal4 enhancer lines, in combination with the live cell markers GFP and DsRed to investigate pupal wing heart development in conjunction with the surrounding tissues. We found that WHPs interact with the tracheal system and specific expression domains of the adult epidermis. Additionally, wing heart development occurs simultaneously with the remodeling of the dorso-lateral epidermis into the scutellum and the scutellar arms. Myogenesis in wing hearts comprises known processes such as founder cell specification, but also new features like removal of growing myotubes, and nuclei movement. Wing heart epithelium development is accomplished by the mesenchymal-epithelial transition of WHPs and occurs slightly delayed to muscle development. The epithelium represents a novel mesodermally derived secondary epithelium. Moreover, we have identified a nerve that runs along the epithelium and innervates the wing heart muscle cells.

Brain anatomy in Diplura (Hexapoda).

BACKGROUND: In the past decade neuroanatomy has proved to be a valuable source of character systems that provide insights into arthropod relationships. Since the most detailed description of dipluran brain anatomy dates back to Hanström (1940) we re-investigated the brains of Campodea augens and Catajapyx aquilonaris with modern neuroanatomical techniques. The analyses are based on antibody staining and 3D reconstruction of the major neuropils and tracts from semi-thin section series. RESULTS: Remarkable features of the investigated dipluran brains are a large central body, which is organized in nine columns and three layers, and well developed mushroom bodies with calyces receiving input from spheroidal olfactory glomeruli in the deutocerebrum. Antibody staining against a catalytic subunit of protein kinase A (DC0) was used to further characterize the mushroom bodies. The japygid Catajapyx aquilonaris possesses mushroom bodies which are connected across the midline, a unique condition within hexapods. CONCLUSIONS: Mushroom body and central body structure shows a high correspondence between japygids and campodeids. Some unique features indicate that neuroanatomy further supports the monophyly of Diplura. In a broader phylogenetic context, however, the polarization of brain characters becomes ambiguous. The mushroom bodies and the central body of Diplura in several aspects resemble those of Dicondylia, suggesting homology. In contrast, Archaeognatha completely lack mushroom bodies and exhibit a central body organization reminiscent of certain malacostracan crustaceans. Several hypotheses of brain evolution at the base of the hexapod tree are discussed.

Pancrustacean phylogeny in the light of new phylogenomic data: support for Remipedia as the possible sister group of Hexapoda.

Remipedes are a small and enigmatic group of crustaceans, first described only 30 years ago. Analyses of both morphological and molecular data have recently suggested a close relationship between Remipedia and Hexapoda. If true, the remipedes occupy an important position in pancrustacean evolution and may be pivotal for understanding the evolutionary history of crustaceans and hexapods. However, it is important to test this hypothesis using new data and new types of analytical approaches. Here, we assembled a phylogenomic data set of 131 taxa, incorporating newly generated 454 expressed sequence tag (EST) data from six species of crustaceans, representing five lineages (Remipedia, Laevicaudata, Spinicaudata, Ostracoda, and Malacostraca). This data set includes all crustacean species for which EST data are available (46 species), and our largest alignment encompasses 866,479 amino acid positions and 1,886 genes. A series of phylogenomic analyses was performed to evaluate pancrustacean relationships. We significantly improved the quality of our data for predicting putative orthologous genes and for generating data subsets by matrix reduction procedures, thereby improving the signal to noise ratio in the data. Eight different data sets were constructed, representing various combinations of orthologous genes, data subsets, and taxa. Our results demonstrate that the different ways to compile an initial data set of core orthologs and the selection of data subsets by matrix reduction can have marked effects on the reconstructed phylogenetic trees. Nonetheless, all eight data sets strongly support Pancrustacea with Remipedia as the sister group to Hexapoda. This is the first time that a sister group relationship of Remipedia and Hexapoda has been inferred using a comprehensive phylogenomic data set that is based on EST data. We also show that selecting data subsets with increased overall signal can help to identify and prevent artifacts in phylogenetic analyses.

A phylogenomic approach to resolve the arthropod tree of life.

Arthropods were the first animals to conquer land and air. They encompass more than three quarters of all described living species. This extraordinary evolutionary success is based on an astoundingly wide array of highly adaptive body organizations. A lack of robustly resolved phylogenetic relationships, however, currently impedes the reliable reconstruction of the underlying evolutionary processes. Here, we show that phylogenomic data can substantially advance our understanding of arthropod evolution and resolve several conflicts among existing hypotheses. We assembled a data set of 233 taxa and 775 genes from which an optimally informative data set of 117 taxa and 129 genes was finally selected using new heuristics and compared with the unreduced data set. We included novel expressed sequence tag (EST) data for 11 species and all published phylogenomic data augmented by recently published EST data on taxonomically important arthropod taxa. This thorough sampling reduces the chance of obtaining spurious results due to stochastic effects of undersampling taxa and genes. Orthology prediction of genes, alignment masking tools, and selection of most informative genes due to a balanced taxa-gene ratio using new heuristics were established. Our optimized data set robustly resolves major arthropod relationships. We received strong support for a sister group relationship of onychophorans and euarthropods and strong support for a close association of tardigrades and cycloneuralia. Within pancrustaceans, our analyses yielded paraphyletic crustaceans and monophyletic hexapods and robustly resolved monophyletic endopterygote insects. However, our analyses also showed for few deep splits that were recently thought to be resolved, for example, the position of myriapods, a remarkable sensitivity to methods of analyses.

Can comprehensive background knowledge be incorporated into substitution models to improve phylogenetic analyses? A case study on major arthropod relationships.

BACKGROUND: Whenever different data sets arrive at conflicting phylogenetic hypotheses, only testable causal explanations of sources of errors in at least one of the data sets allow us to critically choose among the conflicting hypotheses of relationships. The large (28S) and small (18S) subunit rRNAs are among the most popular markers for studies of deep phylogenies. However, some nodes supported by this data are suspected of being artifacts caused by peculiarities of the evolution of these molecules. Arthropod phylogeny is an especially controversial subject dotted with conflicting hypotheses which are dependent on data set and method of reconstruction. We assume that phylogenetic analyses based on these genes can be improved further i) by enlarging the taxon sample and ii) employing more realistic models of sequence evolution incorporating non-stationary substitution processes and iii) considering covariation and pairing of sites in rRNA-genes. RESULTS: We analyzed a large set of arthropod sequences, applied new tools for quality control of data prior to tree reconstruction, and increased the biological realism of substitution models. Although the split-decomposition network indicated a high noise content in the data set, our measures were able to both improve the analyses and give causal explanations for some incongruities mentioned from analyses of rRNA sequences. However, misleading effects did not completely disappear. CONCLUSION: Analyses of data sets that result in ambiguous phylogenetic hypotheses demand for methods, which do not only filter stochastic noise, but likewise allow to differentiate phylogenetic signal from systematic biases. Such methods can only rely on our findings regarding the evolution of the analyzed data. Analyses on independent data sets then are crucial to test the plausibility of the results. Our approach can easily be extended to genomic data, as well, whereby layers of quality assessment are set up applicable to phylogenetic reconstructions in general.