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

Ecdysteroid-induced programmed cell death is essential for sex-specific wing degeneration of the wingless-female winter moth.

The winter moth, Nyssiodes lefuarius, has a unique life history in that adults appear during early spring after a long pupal diapause from summer to winter. The moth exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that cell death of the pupal epithelium of females appears to display condensed chromatin within phagocytes. To provide additional detailed data for interpreting the role of cell death, we performed light microscopy, transmission electron microscopy, and TUNEL assay. We consequently detected two modes of cell death, i.e., dying cells showed both DNA fragmentation derived from epithelial nuclei and autophagic vacuole formation. To elucidate the switching mechanism of sex-specific wing degeneration in females of N. lefuarius, we tested the effects of the steroid hormone 20-hydroxyecdysone (20E) on pupal diapause termination and wing morphogenesis in both sexes. When 20E (5.4 µg) was injected into both sexes within 2 days of pupation, wing degeneration started 4 days after 20E injection in females, whereas wing morphogenesis and scale formation started 6 days after 20E injection in males. We discuss two important findings: (1) degeneration of the pupal wing epithelium of females was not only due to apoptosis and phagocytotic activation but also to autophagy and epithelial cell shrinkage; and (2) 20E terminated the summer diapause of pupae, and triggered selective programmed cell death only of the female-pupal wing epithelium in the wingless female winter moth

TUNEL and DAPI staining of cross sections in pupal wings.

<p>TUNEL assays (green: A, C, E, G) and DAPI staining (cyan: B, D, F, H) of cross sections of female and male pupal wings. (A) Cross section of female pupal wing on Day 4 after the beginning of pupal-adult development. (C) Cross section of female pupal wing on Day 6 after injection of 5.4 µg 20E. (E) Cross section of male pupal wing on Day 7 after beginning of pupal-adult development. (G) Cross section of male pupal wing on Day 8 after injection of 5.4 µg 20E. Note that TUNEL signals (light green) indicated by white arrows were visible in areas of the female wing epithelium. Scale bar  = 50 µm.</p

Induction ratio of 20E application by injection on wing morphogenesis at Day 14 in <i>Nyssiodes lefuarius</i>.

<p>Induction ratio of 20E application by injection on wing morphogenesis at Day 14 in <i>Nyssiodes lefuarius</i>.</p