Molecular Phylogenetics and Evolution 54 (2010) 651–656 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Short Communication On the value of Elongation factor-1a for reconstructing pterygote insect phylogeny Sabrina Simon a,*, Bernd Schierwater a,b, Heike Hadrys a,c a ITZ, Ecology and Evolution, Stiftung Tierärztliche Hochschule Hannover, D-30559 Hannover, Germany b American Museum of Natural History, New York City, NY 10024, USA c Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511, USA article info abstract Article history: Pterygota are traditionally divided in two lineages, the ‘‘Palaeoptera” and Neoptera. Despite several Received 13 May 2009 efforts neither morphology nor molecular systematics have resolved the phylogeny of the pterygote Revised 12 September 2009 insects. Too few markers have yet been identified for adequately tracking mesozoic-aged divergences. Accepted 22 September 2009 We tested the Elongation factor-1a for its phylogenetic value in pterygote insect systematics. This highly Available online 21 October 2009 conserved nuclear protein-coding gene has previously been reported to be useful in other groups for phy- logenetic analyses at the intraordinal level as well as at the interordinal level. The analyses suggest that Keywords: EF-1a DNA sequences as well as intron positions provide informative markers for pterygote Pterygota phylogenetics. Elongation factor-1a Molecular systematics Ó 2009 Elsevier Inc. All rights reserved. EF-1a introns Homologous copies 1. Introduction nuclear coding genes may be promising candidates, since they evolve at a slower rate than mitochondrial coding genes and show The earliest divergence in the evolution of the winged insects little length variability. A few nuclear coding genes have become of formed two lineages, the basal ‘‘Palaeoptera” (insects without wing wider use, including the Elongation factor-1a (EF-1a hereafter) flexion muscles; Odonata and Ephemeroptera,plus severalextinct or- which has proved useful in some terrestrial arthropods (reviewed ders) and the Neoptera (insects with wing flexion muscles). Despite in Caterino et al. (2000)). Several studies have demonstrated that the existence of numerous morphological characters and some large this marker is not only particularly useful for phylogenetic analy- scale molecular data sets the relationships within the Pterygota re- ses among species groups and genera due to variation in silent main ambiguous. The outstanding ‘‘Palaeoptera Problem”, the unre- nucleotide sites (Cho et al., 1995), but also for deeper divergences solved ‘‘comb” among the basal Neoptera (including the positions where amino acid replacements provide phylogenetic information of Dermaptera and Plecoptera) and the origin of the Holometabola (Regier and Shultz, 1997). EF-1a was originally identified as a sin- (insects with complete metamorphosis) are some of the unresolved gle-copy gene in insects. Later it was found that some insect orders branching patterns (Kristensen, 1991; Whitfield and Kjer, 2008). possess multiple copies of EF-1a, as these are Coleoptera, Hyme- So far relatively few molecular markers have been employed to noptera, Diptera, Thysanoptera, Hemiptera – Coccoidae and mem- infer relationships between pterygote orders. The most commonly bers of the Neuropterida (reviewed in Djernæs and Damgaard used molecular markers have been mitochondrial and nuclear (2006)). The paralogs show high nucleotide divergence between rRNA gene fragments (Kjer, 2004; Ogden and Whiting, 2003). For the two functional copies and are considered problematic in high- studying deep splits in insects, nuclear ribosomal genes (18S and er-level phylogenies of insects (Danforth and Ji, 1998; Hovemann 28S) have been the most commonly used molecular markers (e.g. et al., 1988; Jordal, 2002). The identification of a single ortholog Kjer, 2004; Ogden and Whiting, 2003). These have led to concerns in other Hemiptera (von Dohlen et al., 2002), Lepidoptera (Cho about different alignment methods and their effect on resulting et al., 1995) and Odonata (Jordan et al., 2003) suggests indepen- phylogenies (Kjer, 2004; Terry and Whiting, 2005). dent gene duplication in some orders, making EF-1a problematic Consequently, the exploitation of new markers has become a for generic level comparisons but not for phylogenetic studies tar- crucial necessity for resolving pterygote insect phylogeny. Here geting deeper divergence (Goetze, 2006; Lynch and Conery, 2000, 2003). Compared to the often used ribosomal genes the EF-1a gene is much less sensitive to alignment problems while its major disadvantage is that it is relatively short and best suited as a com- * Corresponding author. Address: ITZ, Ecology and Evolution, Stiftung Tierärztli- plement to other, ideally also high quality markers. che Hochschule Hannover, Buenteweg 17d, D-30559 Hannover, Germany. Fax: +49 511 953 8584. In insect systematics Elongation factor-1a has been mostly used E-mail address: [email protected] (S. Simon). for studies within pterygote orders, for example, in Hymenoptera 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.09.029 652 S. Simon et al. / Molecular Phylogenetics and Evolution 54 (2010) 651–656 systematics (Brady and Danforth, 2004), in Coleoptera systematics forming the ML criterion. The assumed model of nucleotide substi- (Jordal, 2002), in Odonata systematics (Groeneveld et al., 2007), for tution was the GTR (general time reversible) model with gamma resolving the phylogeny of the Neuropterida (Haring and Aspöck, distributed rate heterogeneity and an estimated proportion of 2004) and in a combined approach across insect orders (Kjer invariable sites. Support values for ML trees were estimated with et al., 2006). Here we test previous expectations on the value of 1000 bootstrap replicates and 10 random sequence additions per EF-1a for insect systematics at the order level, and demonstrate replicate. its usefulness for inferring phylogenetic relationships among 20 The BI analysis was conducted with MrBayes v.3.1.2 (Huelsen- pterygote insect orders. We focused on a 289 bp coding region beck and Ronquist, 2001) with a GTR + SSR model and a partitioned which is located within the suitable region suggested by Djernæs data set according to the codon positions, allowing independent and Damgaard (2006). These authors argued to focus on a region parameter settings for each partition. The matrix was analyzed between 493 and 1030 according to the mRNA transcript of the over 3000,000 generations using four chains (one cold, three Drosophila melanogaster F1 copy based on their survey of intron heated) and a sampling frequency of 100. Stationarity was evalu- positions and sequenced regions in Hexapoda. These authors fo- ated graphically by plotting log-likelihood scores against genera- cused on the exon–intron structure over a variety of hexapod or- tion and the first 7500 trees were discarded as the ‘‘burn-in”. The ders and the phylogenetic value of these characters. Our purpose remaining trees were assembled into a topology. The sequence of is to evaluate the phylogenetic value based on the nucleotide se- the apterygote Ctenolepisma lineata (Thysanura: GenBank Acces- quence of the recommend EF-1a region including representatives sion No. AF063405) was used in all phylogenetic analyses as the of 20 pterygote orders. The study demonstrates the utility of the outgroup. recommend EF-1a region as molecular marker for phylogenetic analyses among pterygotes even though the amplified region is 3. Results quite short. 3.1. Intron length and positions 2. Materials and methods The isolated central region of the EF-1a gene contained 289 nucleotides of coding sequence plus 55–546 bp of intron sequence Species included in this study are given in Supplementary Mate- across the 40 examined species. According to the mRNA transcript rial. DNA was extracted from ethanol (98%) preserved animals of the Drosophila melanogaster F1 copy (GenBank Accession No. according to a modified standard protocol (Hadrys et al., 1993). X06869) the amplified coding sequences start at position 483 For larger specimens a small tissue from a single leg or alterna- (Fig. 1). The comparative analysis of intron positions reveals some tively wing muscle from the mesothorax was dissected. For smaller clade specific patterns. The longest observed intron contained 546 specimens the entire thorax was used after removing of the gut. A nucleotides (Plec2) and the shortest 55 nucleotides (Mega1), only 289–1039 bp genomic fragment containing the 289 bp coding re- the orders Lepidoptera, Dermaptera, Mecoptera and one Diptera gion of EF-1a was amplified via polymerase chain reaction (PCR) species (Dipt3) had no intron in the amplified region. All others using the primers EF-7 [50-AAC AAR ATG GAY TCN ACN GAR CCN had either one or two introns at two typical intron positions: (a) CC-30] and EF-9 [50-CCN ACN GGB ACH GTT CCR ATA CC-30]. The position 493 (shared by 9 orders) and (b) position 754 (shared by amplification profile was as follows: initial denaturation at 95 °C 14 orders). Only one dipteran species showed an intron in a differ- for 5 min followed by 45 cycles of 30 s at 94 °C, 30 s at 60 °C and ent position (674; Fig. 1). 3 min at 72 °C. PCR products were checked and size determinated by agarose gel electrophoresis. Amplification products from differ- ent species varied in size due to the presence