Incongruent phylogenetic hypotheses and character conflicts in morphology : the root and early branches of the hexapodan tree

Before the turn of the millenium the investigation of phylogenetic relationships was revolutionized by two major inputs, the use of molecular sequence data for phylogenetic reconstruction, paralleled by the sophistication of computer aided reconstruction methods. The ever growing number of data however did not only result in clarifications of open questions, but brought forth a number of new conflicting phylogenetic hypotheses. Sometimes they are wrongly referred to as conflicts between morphological and molecular approaches, which sporadically even culminated in the rejection of the usefulness of one of the two approaches (e.g. Scotland et al 2003). These scientists overlook the great advantage of having two a priori largely independent data sets (Wägele 2001) which in a synthetic way enable the greatest progress in phylogenetic research. However, solely putting data together will not suffice to choose among conflicting hypotheses. The increasing number of conflicts necessitates approaches that go beyond mere data congruence, but searching for the possible reasons of conflicts. In the present paper, problems in the reconstruction of the phylogenetic origin of Hexapoda, as well as of the early branchings within the Hexapoda, will exemplify approaches of critical re-evaluation and testing of data used in morphological data matrices for phylogenetic analyses. The early cladogenetic events of hexapods are especially suited for such a discussion for several reasons. The hexapods, as the most species-rich group of organisms, look back at a long and multi-faceted history of taxonomic and phylogenetic studies, culminating in a number of conflicting hypotheses. Triggered by incongruences with morphological analyses the reconstruction of the hexapodan roots likewise became a hot-spot of molecular research activities during^the last two decades. Furthermore the phylogenetic positions of the oldest lineages branching off within the hexapodan clade, the Diplura, Protura and Collembola, are in particular very difficult to reconstruct. While at least the latter two are well defined by morphological autapomorphies their phylogenetic position could not be reconstructed unambiguously, since their morphology seems highly derived with respect to the hexapodan ground pattern.Angestoßen durch molekulare Untersuchungen sind die Verwandtschaftsverhältnisse der Großgruppen der Arthropoden in der letzten Dekade ein Hotspot der phylogenetischen Diskussion geworden. Ein heiß diskutiertes Thema ist die Frage nach der Monophylie der Insekten. Die in diesem Zusammenhang besonders interessanten „Ur-Insekten“ bilden eine morphologisch sehr heterogene Gruppe von primär flügellosen Insekten. Während die Annahme verwandtschaftlicher Beziehungen der Archaeognatha und Zygentoma zu den Pterygota (Ectognatha) breite Unterstützung finden, gibt es widersprüchliche Hypothesen hinsichtlich der phylogenetischen Stellung der Collembola, Protura und Diplura. Zu heftigen Diskussionen haben die Analysen mitochondrialer Gene durch Nardi et al. (2003) geführt. In den rekonstruierten Bäumen sind die Ectognatha näher verwandt mit Crustaceen als mit Collembolen, wodurch die Hexapoden eine paraphyletische Gruppierung werden. Dieser Befund hat eine Flut von neuen molekularen Untersuchungen ausgelöst und nahezu alle denkbaren Kombinationen der Verwandtschaftsbeziehungen zwischen Collembola, Protura, Diplura und Ectognatha haben Unterstützung gefunden. Kann die Morphologie zur Klärung dieser phylogenetischen Konflikte entscheidend beitragen? Sind die Hexapoden wirklich ein Monophylum oder ist die Hexapodie mehrfach unabhängig voneinander entstanden? Nach Diskussion ausgewählter Konflikte zur phylogenetischen Stellung der verschiedenen „Ur-Insekten“ werden anhand eigener Untersuchungen exemplarisch einige Probleme morphologischer Datensätze in der modernen stammesgeschichtlichen Forschung aufgezeigt und entsprechende Lösungsmöglichkeiten diskutiert

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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

BackgroundPhylogenetic 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.ResultsWe 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.ConclusionsOur 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

Gene content evolution in the arthropods

Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

Application of propylene glycol in DNA-based studies of invertebrates

High-throughput sequencing (HTS) studies on invertebrates commonly use ethanol as the main sample fixative (upon collection) and preservative (for storage and curation). However, alternative agents exists, which should not be automatically neglected when studies are newly designed. This review provides an overview of the application of propylene glycol (PG) in DNA-based studies of invertebrates, thus to stimulate an evidence-based discussion. The use of PG in DNA-based studies of invertebrates is still limited (n = 79), but a steady increase has been visible since 2011. Most studies used PG as a fixative for passive trapping (73%) and performed Sanger sequencing (66%; e.g. DNA barcoding). More recently, HTS setups joined the field (11%). Terrestrial Coleoptera (30%) and Diptera (20%) were the most studied groups. Very often, information on the grade of PG used (75%) or storage conditions (duration, temperature) were lacking. This rendered direct comparisons of study results difficult, and highlight the need for further systematic studies on these subjects. When compared to absolute ethanol, PG can be more widely and cheaply acquired (e.g. as an antifreeze, 13% of studies). It also enables longer trapping intervals, being especially relevant at remote or hard-to-reach places. Shipping of PG-conserved samples is regarded as risk-free and is authorised, pinpointing its potential for larger trapping programs or citizen science projects. Its property to retain flexibility of morphological characters as well as to lead to a reduced shrinkage effect was especially appraised by integrative study designs. Finally, the so far limited application of PG in the context of HTS showed promising results for short read amplicon sequencing and reduced representation methods. Knowledge of the influence of PG fixation and storage for long(er) read HTS setups is currently unavailable. Given our review results and taking difficulties of direct methodological comparisons into account, future DNA-based studies of invertebrates should on a case-by-case basis critically scrutinise if the application of PG in their anticipated study design can be of benefit

The unexpected diverse venom evolution in centipedes:

Jüngste Erkenntnisse aus der modernen Tiergiftforschung zeigen, dass die Vorfahren der Hundertfüßer vermutlich ein recht simples Gift mit wenigen neurotoxischen Komponenten und Enzymen besaßen. Dieser Giftcocktail wurde im Laufe der Evolution unerwartet stark verändert und an die heutige Lebensweise der Hundertfüßer angepasst. Tatsächlich gibt es keine Toxinfamilie, die in den Giften der untersuchten Arten aller fünf Ordnungen gemeinsam vorkommt, und damit gibt es auch nicht das eine Hundertfüßergift. Die Klimax sind die extrem diversen Gifte der Skolopender, die zahlreiche neue Neurotoxine beinhalten. Die Prozesse, wie die Giftproteine in den Hunderfüßern entstehen und evolvieren, sind hier durch neue Studien beleuchtet. Erste Daten zeigen, dass auf genomischer Ebene sogar horizontaler Gentransfer eine Rolle spielen könnte.Recent studies in the field of modern venomics illustrate that the ancestors of today’s centipedes presumably had a rather simple venom cocktail at their disposal with few neurotoxic components and enzymes. In the course of evolution, this venom cocktail has experienced unexpected changes with diverse adaptations to centipede lifestyles – a development that led to unique venoms in all five centipede orders that share no single toxin family, which makes it impossible to speak of a centipede venom. The venoms of giant centipedes (Scolopendromorpha) represent a climax with several new neurotoxic peptides. The processes that have led to the development and evolution of venom proteins in centipedes are illustrated here. First data on genomic level suggest that even horizontal gene transfer might play an important role

100 years of research on the Protura: many secrets still retained

The Protura were discovered relatively late in the history of entomology. The first description of these minute soil arthropods was given in 1907 by the Italian entomologist Filippo Silvestri, who named them ‘Protura’. Shortly thereafter his fellow countryman Antonio Berlese published two brief notes on these animals before his grand monograph of the ‘Myrientomata’, as he named them, appeared in 1909. The centennial of the discovery of Protura offers the opportunity to review our knowledge about these peculiar animals. In the end, we must confess that proturans continue to retain an amazing number of secrets, including basic facts of knowledge about their biology and ecology. Up to the present, they have concealed from scientific observation their sexual life and the mode of sperm transmission. For a long time, their egg deposition and early development were completely obscure topics; only recently has it become possible to conduct first observations on their embryology. The list of open questions can be easily extended: we know only little about their nutritional biology, and next to nothing about their sensory systems, communication, physiological and ecological capacities. The greatest progress in proturan research over the past 100 years has been made in the field of taxonomy. Nonetheless, the road of taxonomy was bumpy and the proturans proved to be awkward travel companions. Species are exceedingly difficult to determine; and the number of living scientists that are able to unambiguously identify specimens at the species level can be counted on two hands. Proturans inhabit soils in all terrestrial regions of the earth (excepting the Arctic and Antarctic regions). Presently, a total of 787 valid species has been described. Because of the high standards of Berlese’s 1909 monograph, several decades would pass before morphological and anatomical research could equal or go beyond his classical landmark. In more recent times, remarkable contributions have been made to the ultrastructural investigation of internal organs and sperm morphology; hereby, the proturans proved again to be odd and unusual in many respects. The peculiarities of Protura have evoked heated debates about their phylogenetic position from the beginning, and such discussions have recently been revived by the introduction of molecular data. The review comprehends an extensive reference list on all research topics except taxonomy, which recently was compiled by Szeptycki (2007)

DNA Barcoding of <i>Trichobilharzia</i> (Trematoda: Schistosomatidae) Species and Their Detection in eDNA Water Samples

We designed and tested species-specific PCR primers to detect Trichobilharzia species via environmental DNA (eDNA) barcoding in selected Austrian water bodies. Tests were performed with eDNA samples from the field as well as with artificial samples from the lab, where snails releasing cercariae were kept in aquariums. From two localities, Trichobilharzia was documented based on the release of cercariae from snails, enabling morphological species identification. In both cases, the corresponding species were detected via eDNA: Trichobilharzia szidati and Trichobilharzia physellae. Nonetheless, the stochasticity was high in the replicates. PCR tests with aquarium water into which the cercariae had been released allowed eDNA detection even after 44 days. As in the PCRs with eDNA samples from the field, positive results of these experiments were not obtained for all samples and replicates. PCR sensitivity tests with dilution series of T. szidati genomic DNA as well as of PCR amplification products yielded successful amplification down to concentrations of 0.83 pg/µL and 0.008 pg/µL, respectively. Our results indicate that the presumed species specificity of PCR primers may not be guaranteed, even if primers were designed for specific species. This entails misidentification risks, particularly in areas with incomplete species inventories

DellAmpio_et_al_2013: Supplementary Data

Supplementary File Archives of "Decisive Datasets in Phylogenomics: Lessons from Studies on the Phylogenetic Relationships of Primarily Wingless Insects