The mitochondrial genome of Bacillus stick insects (Phasmatodea) and the phylogeny of orthopteroid insects Federico Plazzi ⇑ , Andrea Ricci, Marco Passamonti Department of Biologia Evoluzionistica Sperimentale, University of Bologna, via Selmi 3, 40126 Bologna, Italy article info Article history: Received 28 July 2010 Revised 6 December 2010 Accepted 11 December 2010 Available online 16 December 2010 Keywords: mtDNA Phasmatodea Bacillus Phylogeny Phylogenetic informativeness abstract The Order Phasmatodea (stick and leaf insects) includes many well-known species of cryptic phytopha- gous insects. In this work, we sequenced the almost complete mitochondrial genomes of two stick insect species of the genus Bacillus. Phasmatodea pertain to the Polyneoptera, and represent one of the major clades of heterometabolous insects. Orthopteroid insect lineages arose through rapid evolutionary radi- ation events, which likely blurred the phylogenetic reconstructions obtained so far; we therefore per- formed a phylogenetic analysis to resolve and date all major splits of orthopteroid phylogeny, including the relationships between Phasmatodea and other polyneopterans. We explored several molec- ular models, with special reference to data partitioning, to correctly detect any phylogenetic signal lying in rough data. Phylogenetic Informativeness analysis showed that the maximum resolving power on the orthopteroid mtDNA dataset is expected for the Upper Cretaceous, about 80 million years ago (Mya), but at least 70% of the maximum informativeness is also expected for the 150–200 Mya timespan, which makes mtDNA a suitable marker to study orthopteroid splits. A complete chronological calibration has also been computed following a Penalized Likelihood method. In summary, our analysis confirmed the monophyly of Phasmatodea, Dictyoptera and Orthoptera, and retrieved Mantophasmatodea as sister group of Phasmatodea. The origin of orthopteroid insects was also estimated to be in the Middle Triassic, while the order Phasmatodea seems to appear in the Upper Jurassic. The obtained results evidenced that mtDNA is a suitable marker to unravel the ancient splits leading to the orthopteroid orders, given a proper methodological approach. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Insects (Insecta) are among the most diverse and successful ter- restrial organisms, showing a great variety of shapes and life hab- its. Commonly, they are subdivided into two main lineages: Palaeoptera and Neoptera. The monophyly of Palaeoptera, which comprise, among the others, ephemerids, dragonflies and damsel- flies, has been sometimes contentious (see Wheeler et al., 2001; Whitfield and Kjer, 2008; and references therein), while Neoptera are always acknowledged as a monophyletic taxon (Wheeler et al., 2001; and references therein). Among neopteran insects, Martynov (1925) first introduced a group named Polyneoptera, further partitioned into Blattopteroi- dea (nowadays known as Dictyoptera) and Orthopteroidea. The Polyneoptera, collectively referred to as ‘‘orthopteroid insects’’ (Bradler, 2009; Terry and Whiting, 2005; Wheeler et al., 2001), are the outcome of an ancient evolutionary radiation, leading to a heterogeneous assemblage, displaying many forms and adapta- tions, and about one third of the total insect diversity at the ordinal level (Terry and Whiting, 2005). They include Blattodea (roaches), Dermaptera (earwigs), Embiidina (web-spinners), Grylloblattodea (ice crawlers), Isoptera (termites), Mantodea (praying mantises), Orthoptera (grasshoppers and crickets), Plecoptera (stoneflies), Zoraptera (angel insects), and Phasmatodea (stick and leaf insects). Recently, a new polyneopteran order has been discovered and named Mantophasmatodea (gladiators) (Klass et al., 2002; Zompro, 2001). Although the monophyly of Polyneoptera is widely acknowledged by most studies (Bradler, 2009; Grimaldi and Engel, 2005; Gullan and Cranston, 2005; Wheeler et al., 2001; Willmann, 2004), others do not accept it (see Haas and Kukalová-Peck, 2001; and references therein); moreover, molecular data do not always retrieve Polyneoptera as monophyletic (Cameron et al., 2006a; Kjer, 2004; Kjer et al., 2006; Terry and Whiting, 2005; Whitfield and Kjer, 2008; Whiting, 2002). Phylogenetic relationships within Polyneoptera are also quite controversial (Bradler, 2009; Flook and Rowell, 1998; Ishiwata et al., 2010; Wheeler et al., 2001; and references therein). 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.12.005 Abbreviations: AIC, Akaike Information Criterion; BF, Bayes Factor; EML, Estimated Marginal Likelihood; ML, Maximum Likelihood; mtDNA, mitochondrial DNA; Mya, million years ago; Myr, million years; PCG, protein-coding gene; PL, Penalized Likelihood. ⇑ Corresponding author. Fax: +39 051 2094286. E-mail address: federico.plazzi@unibo.it (F. Plazzi). Molecular Phylogenetics and Evolution 58 (2011) 304–316 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev