Arthropod Structure & Development 39 (2010) 88–110 Contents lists available at ScienceDirect Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd Arthropod phylogeny revisited, with a focus on crustacean relationships Stefan Koenemann a,*, Ronald A. Jenner b,**, Mario Hoenemann a, Torben Stemme a, Bjo¨rn M. von Reumont c,*** a Institute for Animal Ecology and Cell Biology, University of Veterinary Medicine Hannover, Bu¨nteweg 17d, D-30559 Hannover, Germany b Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK c Zoologisches Forschungsmuseum Alexander Koenig, Adenauerallee 160, 53113 Bonn, Germany article info abstract Article history: Higher-level arthropod phylogenetics is an intensely active field of research, not least as a result of the Received 10 June 2009 hegemony of molecular data. However, not all areas of arthropod phylogenetics have so far received Accepted 14 October 2009 equal attention. The application of molecular data to infer a comprehensive phylogeny of Crustacea is still in its infancy, and several emerging results are conspicuously at odds with morphology-based studies. Keywords: In this study, we present a series of molecular phylogenetic analyses of 88 arthropods, including 18S rDNA 57 crustaceans, representing all the major lineages, with Onychophora and Tardigrada as outgroups. Our 16S rDNA analyses are based on published and new sequences for two mitochondrial markers, 16S rDNA and Cytochrome c oxidase subunit I Structural alignment cytochrome c oxidase subunit I (COI), and the nuclear ribosomal gene 18S rDNA. We designed our Multiple alignment optimization phylogenetic analyses to assess the effects of different strategies of sequence alignment, alignment Sensitivity analysis masking, nucleotide coding, and model settings. Our comparisons show that alignment optimization of ribosomal markers based on secondary structure information can have a radical impact on phylogenetic reconstruction. Trees based on optimized alignments recover monophyletic Arthropoda (excluding Onychophora), Pancrustacea, Malacostraca, Insecta, Myriapoda and Chelicerata, while Maxillopoda and Hexapoda emerge as paraphyletic groups. Our results are unable to resolve the highest-level relation- ships within Arthropoda, and none of our trees supports the monophyly of Myriochelata or Mandibulata. We discuss our results in the context of both the methodological variations between different analyses, and of recently proposed phylogenetic hypotheses. This article offers a preliminary attempt to incor- porate the large diversity of crustaceans into a single molecular phylogenetic analysis, assessing the robustness of phylogenetic relationships under varying analysis parameters. It throws into sharp relief the relative strengths and shortcomings of the combined molecular data for assessing this challenging phylogenetic problem, and thereby provides useful pointers for future studies. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction groups, Hexapoda, Myriapoda, Crustacea, Chelicerata, and the extinct Trilobitomorpha, has remained a matter of debate since the 19th One of the persistent challenges in systematic biology concerns century (e.g., Latreille, 1817; Pocock, 1893a,b; Lankester, 1904). the phylogenetic relationships of Arthropoda (here defined as Although debates about arthropod phylogeny have long been framed all extant arthropods and their stem groups, but excluding in terms of morphological and developmental evidence, current onychophorans and tardigrades; Panarthropoda refers to the activities show an additional strong focus on molecular data, derived grouping of Arthropoda, Tardigrada and Onychophora). The system- from both mitochondrial and nuclear sources. In this article, we atic literature on higher-level relationships within arthropods dwarfs present a series of molecular phylogenetic analyses of arthropod that of any metazoan taxon, with the possible exception of verte- phylogeny based on both published and new sequence data from brates. The phylogenetic relationships of the five major traditional three loci: 18S rDNA, 16S rDNA, and cytochrome c oxidase I (COI). We discuss our results with respect to recent phylogenetic analyses based on molecular and morphological evidence. The specific focus of * Corresponding author. Tel.: þ49 511 953 8881. our analyses is the relationships between the major lineages of ** Corresponding author. Tel.: þ44 207 942 6885. crustaceans, which representoverhalf of the species in ourdata set. To *** Corresponding author. Tel.: þ49 228 912 2351. E-mail addresses: [email protected] (S. Koenemann), our knowledge our data set includes the largest sample of crustacean [email protected] (R.A. Jenner), [email protected] (B.M. von Reumont). diversity yet analyzed in a single molecular phylogenetic analysis. 1467-8039/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2009.10.003 S. Koenemann et al. / Arthropod Structure & Development 39 (2010) 88–110 89 1.1. The modern fate of early phylogenetic concepts or combined molecular and morphological evidence. Our current understanding of the phylogenetic positions and evolution of Several of the familiar higher-level groupings, such as Atelo- extinct panarthropod lineages is of course wholly dependent on the cerata, Tracheata, Uniramia, and Mandibulata, have their origins as deployment of morphological data (Vaccari et al., 2004; Cobbett far back as the 19th century. For example, Haeckel (1866) erected et al., 2007). Not least, excellent morphological work on fossils has Tracheata, to which he assigned all arthropods with tracheal allowed unique insights into the composition of stem-lineages that breathing, the arachnids, myriapods and insects. Tracheata was underpin the extant crown groups of panarthropods (e.g., Walossek redefined by Pocock (1893a,b), who excluded the arachnids. Pocock and Mu¨ ller, 1990; Walossek and Mu¨ ller, 1998; Walossek, 1993; furthermore considered Myriapoda ‘‘an unnatural assemblage of Budd, 1996; Edgecombe, 2004). beings’’, composed of diplopods/pauropods and chilopods/hexa- pods as the two most closely related groups, and symphylans in an 1.2. Modern debates and molecular evidence unassigned position (‘‘a question for future discussion’’). Based on a detailed comparison of metameric structures, Heymons (1901) The application of diverse and increasingly abundant molecular continued to support myriapods and hexapods as sister groups, and evidence to the problem of higher-level arthropod phylogeny is proposed to unite them under the new name Atelocerata. Today, currently gathering steam on several fronts. First, commonly used both concepts, Tracheata and Atelocerata, are usually used as nuclear and mitochondrial loci, including 18S rDNA, 28S rDNA, 16S synonyms. Interestingly, in the phylogenetic analysis of combined rDNA, COI, elongation factor 1-a, and RNA polymerase II are molecular and morphological evidence of Wheeler et al. (2004), sequenced for increasing numbers of species across all major extant a monophyletic Atelocerata is supported depending on whether panarthropod taxa. Second, studies that go beyond these ‘‘usual selected fossils are included in the analysis. Molecular analyses do suspects’’ have started to add valuable independent light on not find support for Atelocerata, instead uniting Crustacea and arthropod relationships (Regier et al., 2005, 2008). Third, the Hexapoda as Pancrustacea (e.g., Regier and Shultz, 1997) or Tetra- development of increasingly sophisticated and powerful conata (Dohle, 2001). sequencing and computational techniques, and the rapidly falling Some early hypotheses about the evolutionary relationships of prices of large-scale sequencing will soon take arthropod phylo- arthropods included other segmented animals, such as onycho- genetics to the same level as higher-level metazoan phylogenetics. phorans, as basal arthropods, from which modern, extant forms Our study contributes to the first category by analyzing potential were believed to have been derived (e.g., Snodgrass, 1935, 1938). phylogenetic signal in a combined data set of three oft-used loci Manton (1973) went a step further and proposed the taxon Uni- (18S rDNA, 16S rDNA, COI), and goes beyond previous efforts by ramia to embrace hexapods, myriapods, and onychophorans, three (1) an increased sampling of taxa within Crustacea, (2) the use of groups characterized by segmented trunks, single-branch limbs, newly developed software to improve the quality of multiple one pair of (first) antennae, and reduced post-oral mouthparts. She sequence alignments, and (3) the performance of sensitivity ana- considered Crustacea, Chelicerata, and Trilobita to be separate lyses to explore the effect of differences in sequence alignment on groups from each other and from Uniramia. However, despite the phylogenetic results. apparent support from neuroanatomical data for including A large number of molecular phylogenetic analyses of major Onychophora within Arthropoda (Strausfeld et al., 2006), the arthropod relationships (some also including morphological data) Uniramia hypothesis is now generally considered obsolete (see has been published, but despite some emerging consensus many Wa¨gele, 1993). Nevertheless, a recent molecular phylogenetic unresolved issues remain (e.g., Giribet, et al., 1996; Fortey and analysis (Colgan et al., 2008) places Onychophora within Arthro- Thomas, 1997; Wheeler, 1997; Zrzavy et al., 1997; Giribet et al., poda,