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

https://doi.org/10.1016/j.asd.2018.05.002Get rights and content

Highlights

  • The early branching events among winged insects are reassessed.

  • Emphasis was put on evaluation of confounding signal in phylogenomic datasets.

  • Metapterygota excluded as a scenario of early winged insect evolution.

  • Phylogenetic signal remains ambiguous: Palaeoptera and Chiastomyaria are supported.

  • Morphologically, thorax and abdomen reanalyzes warranted.

Abstract

The phylogenetic relationships of the winged insect lineagesmayflies (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.

Introduction

The phylogenetic relationships between the three lineages mayflies (Ephemeroptera), damselflies and dragonflies (Odonata), and all other winged insects (Neoptera), subsumed as the “Palaeoptera Problem” (Hovmöller et al., 2002), are a matter of debate now for decades. Resolving these early branching events within pterygote evolution has been a persisting challenge using molecular data (Whiting et al., 1997, Wheeler, 2001, Hovmöller et al., 2002). Also a wealth of morphological arguments have been brought forward in support of the three different hypotheses on phylogenetic relationships. Here, we only briefly state these morphological arguments, detailed overviews have been given in Klass (2007) and Klass (2009), and highlight various molecular studies.

The morphological arguments in favor of the Chiastomyaria hypothesis (Ephemeroptera + Neoptera; Fig. 1a) are based on the direct sperm transfer in Ephemeroptera and all neopteran insects. Compared to Chiastomyaria, Odonata have a male secondary copulatory apparatus which functions as a temporary sperm reservoir, sperm removal, and sperm transfer system (Tillyard, 1917, Boudreaux, 1979, Corbet, 1980, Pfau, 1991, Pfau, 2011). Also, the similar functioning of the flight apparatus of Ephemeroptera and Neoptera with thoracic muscle groups attached to the tergum and thus moving the wings indirectly during the upstroke was proposed as a potential synapomorphy (Matsuda, 1970, Pfau, 1986, Pfau, 1991).

Molecular support for Chiastomyaria was mainly provided by analyses of nuclear rRNA data (18S and/or 28S) based on improved alignment procedures (Kjer, 2004, Yoshizawa and Johnson, 2005, Misof et al., 2007) and biologically more realistic alignments based on RNA secondary structures (Mallatt and Giribet, 2006, von Reumont et al., 2009). Also analyses of large-scale datasets e.g. within mitogenomic studies (Wan et al., 2012, Li et al., 2014) and ‘phylogenomic’ studies – multi-gene approaches based on whole genome and/or transcriptomic data (Simon et al., 2009, Letsch and Simon, 2013) supported Chiastomyaria.

Metapterygota (Odonata + Neoptera; Fig. 1b) was proposed based on features of the mandibular food uptake system, the respiratory system, the imaginal molts and the circulatory system (Staniczek, 2000, Staniczek, 2001, Beutel and Pohl, 2006, Pass, 2006). In Odonata and Neoptera, the mandibles are anteriorly connected to the head with a ball-and-socket joint whereas in Ephemeroptera an anterior articulation complex is present which is in fact composed of two joints, an anterolateral and a paratentorial part (Staniczek, 2000, Staniczek, 2001). A homologous anterior articulation complex is also present in Zygentoma, the sistergroup of all winged insects (Staniczek, 2000, Staniczek, 2001, Blanke et al., 2014, Blanke et al., 2015). Concerning the respiratory system, the meso- and metathoracic legs and wings are supplied by two tracheal stems each originating from the spiraculi of the same and the following segment in Odonata and Neoptera (Kristensen, 1975, Kristensen, 1981). Moreover, Ephemeroptera are the only winged insects, which undergo an additional molt during the winged stage.

Molecular support for Metapterygota is comparably low (e.g., Whiting et al., 1997, Wheeler et al., 2001, Hovmöller et al., 2002, Ogden and Whiting, 2003) and mainly driven by mitogenomic approaches (Ma et al., 2014, Song et al., 2016). Simon et al. (2012) recovered Metapterygota based on a phylogenomic approach but it was also highlighted that phylogenomic data matrices have more complex phylogenetic signal, thus recovering different branching scenarios.

Palaeoptera (Odonata + Ephemeroptera; Fig. 1c) was proposed based on similarities in the antennae, maxillae, and wings (Brauckmann and Zessin, 1989, Bechly et al., 2001, Kukalová-Peck, 2008). In Odonata and Ephemeroptera the pedicellus is longer than the scapus, and both taxa lack antennal circulatory organs in the adult stage. The maxillae in both taxa are characterized by the presence of mesally directed dentisetae, and strengthened setae-like structures at the lacinia (Staniczek, 2000, Staniczek, 2001, Blanke et al., 2012). However, it has to be emphasized that subapical rows of setae superficially resembling dentiseta-like structures were also shown for Blattodea (Zhuzhikov, 2007), but at present it is unclear from the published data, whether the depicted mesal rows of setae really constitute dentisetae. Further investigation on a larger taxon sampling with more detailed documentation of the lacinial morphology is necessary in order to derive a well-informed conclusion about the identity of these structures.

Furthermore, a plethora of wing characters has been proposed as synapomorphic for Odonata and Ephemeroptera: the inability to fold the wings over the abdomen horizontally, similar wing-base sclerites and similarities in the intercalary veins, the proximate stem of the anterior radial vein (RA) and the posterior radial vein (RP) as well as the common stem of anterior medial vein (MA) and posterior medial vein (MP) (Riek and Kukalová-Peck, 1984, Willkommen and Hörnschemeyer, 2007, Yoshizawa and Ninomiya, 2007, Kukalová-Peck, 2008).

The Palaeoptera hypothesis was predominantly supported by earlier molecular studies and nuclear rRNA data analyses (18S and/or 28S). Ogden and Whiting (2003) demonstrated that the particular arrangement of Odonata and Ephemeroptera is extraordinarily sensitive to alignment methodology, the alignment parameters selected within a particular methodology, and the method of tree reconstruction. As a consequence support increased for Chiastomyaria (see above), or, alternatively, Palaeoptera was only weakly supported (Letsch et al., 2010). Alternative molecular markers, such as nuclear protein coding genes (DNA polymerase delta) and the two largest subunits of RNA polymerase II (Ishiwata et al., 2010, Sasaki et al., 2013) or a combination of nuclear rRNA data (18S and 28S) and nuclear protein coding genes (Histone 3 and Elongation factor-1α), again supported the Palaeoptera hypothesis (Kjer et al., 2006). The Palaeoptera hypothesis also gained increased support by several phylogenomic studies (e.g., Meusemann et al., 2010, von Reumont et al., 2012). However, Thomas et al. (2013) showed the impact of taxon sampling, outgroup choice and heterogeneous base composition across taxa on the retrieved relationships between Odonata, Ephemeroptera and Neoptera. Their analyses of datasets based on Sanger sequencing data including nuclear rRNA data (12S, 18S and 28S) and nuclear and mitochondrial protein coding genes (Histone 3, Elongation factor-1α and the mitochondrial gene cytochrome oxidase subunit II) consistently supported Palaeoptera. Moreover, they reanalyzed previously published mitogenomic and phylogenomic studies and highlighted potential sources of systematic error. A recent phylogenomic study addressing the relationships among all major insect lineages supported Palaeoptera in all tree reconstructions (Misof et al., 2014). However, analyses of alternative signal using Four-cluster Likelihood Quartet Mapping also revealed inherent phylogenetic signal for Chiastomyaria, which is not visible from simple tree reconstruction analyses alone.

To further shed light on the early branching events within pterygote evolution, we reanalyzed the Palaeoptera problem in an attempt to address potential alternative and/or confounding signal within phylogenomic data. We therefore compiled new datasets mainly based on transcriptomes published by Misof et al. (2014) and including data from whole genomes. In addition, we evaluated in-depth confounding and alternative signal using various methodological approaches. In addition, the morphological arguments brought forward since the reviews of Klass (2007) and Klass (2009) are discussed in the light of results we obtained from our molecular analyses.

Section snippets

Data set generation

We compiled two different datasets (Supplementary Material Table S1). Dataset A comprised 41 taxa, including sequence data from five Odonata and five Ephemeroptera species, seven primarily wingless Hexapoda (two Diplura, two Archaeognatha and three Zygentoma), and one representative each for the 24 currently recognized neopteran insect orders. Data were derived from transcriptomes or genomes. In dataset B, we included only neopteran taxa for which (draft) genomes were available, leading to 26

The early branching events of winged insects

Palaeoptera was inferred in all analyses with 100% bootstrap support in both datasets (Fig. 2 and Supplementary Material Fig. S3). Other relationships, except for a few clades within polyneopteran and paraneopteran insects in the tree derived from dataset A, were maximally supported. Zygentoma was inferred as sistergroup to Pterygota in all trees with maximal support (Fig. 2, Supplementary Material Fig. S3). The high support for Palaeoptera is in agreement with most molecular multi-gene or

Conclusion

In contrast to earlier molecular studies, which also supported Metapterygota, the present study excludes this hypothesis as a possible evolutionary scenario of early winged insects. However, ambiguities still exist concerning the Palaeoptera and Chiastomyaria hypotheses. Besides methodological considerations concerning systematic bias, the comparatively low coverage of Ephemeroptera and primarily wingless Hexapoda might affect current phylogenetic reconstructions. However, simply adding more

Acknowledgements

The authors thank Panagiotis Provataris (ZMB, ZFMK Bonn, Germany), Bernhard Misof (ZMB, ZFMK Bonn, Germany) and Stephen Richards (Baylor, Houston, USA) in the frame of the i5K consortium for granting us permission to use and analyze the unpublished genome sequence and preliminary official gene sets of the dragonfly Ladona fulva and the mayfly Ephemera danica. The authors also especially thank the 1KITE Odonata group (www.1kite.org/subprojects.html), Ryuichiro Machida (University of Tsukuba,

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