Molecular Phylogenetics and Evolution 79 (2014) 169–178 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Towards a mitogenomic phylogeny of Lepidoptera ⇑ Martijn J.T.N. Timmermans a,b, , David C. Lees c, Thomas J. Simonsen a a Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom b Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom c Department of Zoology, Cambridge University, Downing Street CB2 3EJ, United Kingdom article info abstract Article history: The backbone phylogeny of Lepidoptera remains unresolved, despite strenuous recent morphological and Received 13 January 2014 molecular efforts. Molecular studies have focused on nuclear protein coding genes, sometimes adding a Revised 11 May 2014 single mitochondrial gene. Recent advances in sequencing technology have, however, made acquisition of Accepted 26 May 2014 entire mitochondrial genomes both practical and economically viable. Prior phylogenetic studies utilised Available online 6 June 2014 just eight of 43 currently recognised lepidopteran superfamilies. Here, we add 23 full and six partial mitochondrial genomes (comprising 22 superfamilies of which 16 are newly represented) to those Keywords: publically available for a total of 24 superfamilies and ask whether such a sample can resolve deeper tRNA rearrangement lepidopteran phylogeny. Using recoded datasets we obtain topologies that are highly congruent with Ditrysia Illumina prior nuclear and/or morphological studies. Our study shows support for an expanded Obtectomera LR-PCR including Gelechioidea, Thyridoidea, plume moths (Alucitoidea and Pterophoroidea; possibly along with Pooled mitochondrial genome assembly Epermenioidea), Papilionoidea, Pyraloidea, Mimallonoidea and Macroheterocera. Regarding other controversially positioned higher taxa, Doidae is supported within the new concept of Drepanoidea and Mimallonidae sister to (or part of) Macroheterocera, while among Nymphalidae butterflies, Danainae and not Libytheinae are sister to the remainder of the family. At the deepest level, we suggest that a tRNA rearrangement occurred at a node between Adeloidea and Ditrysia + Palaephatidae + Tischeriidae. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction includes a number of biological model organisms, many severe pest species, and comprises many of the best known and most Reconstructing a stable phylogenetic framework for Lepidop- popular invertebrates, emphasising that studies into lepidopteran tera at the superfamily level is an ongoing process that, despite phylogeny and evolution are of both scientific and public interest. considerable effort over recent years, is still far from complete. It is therefore not surprising that the past few years have seen sev- Kristensen and Skalski (1999) overview summarised the most eral attempts to resolve the higher-level phylogeny of Lepidoptera comprehensive morphology-based hypothesis of the last century based on comprehensive molecular datasets. The ‘‘LepTree project’’ for the phylogeny of the order. While the early part of Lepidoptera has already resulted in a number of benchmark publications evolution was presented as a well-resolved ‘‘Hennigean comb’’, the focused both at the superfamily level (e.g. Regier et al., 2009, phylogeny of the vast majority of the order (the suborder Ditrysia 2013; Cho, et al. 2011; Bazinet et al., 2013; Sohn et al., 2013) and which comprise more than 95% of all known species) was almost for clusters of several larger superfamilies (Zwick et al., 2011; entirely unresolved apart from a few higher-level clades. This Regier et al., 2012a,b). The ‘‘Ditrysia project’’ has so far resulted overview made it abundantly clear that large-scale molecular in a superfamily-level molecular phylogeny (Mutanen et al., analyses were needed to resolve the higher-level phylogeny of 2010) and a phylogeny of Papilionoidea (Heikkila et al., 2012), ditrysian Lepidoptera. while associated studies have focused on the superfamilies With more than 157,000 described species (van Nieukerken Gelechioidea (Kaila et al., 2011) and Noctuoidea (Zahiri et al., et al., 2011), Lepidoptera (butterflies and moths) represent one of 2011, 2012, 2013a,b), as well as the large family Gelechiidae the evolutionarily most successful lineages of phytophagous (Karsholt et al., 2013). Both the LepTree project and the Ditrysia insects (e.g. Grimaldi and Engel, 2005). Furthermore, the order project have provided considerable new resolution to the higher phylogeny of Ditrysia, but many groupings have been mutually exclusive between the studies and others were not well supported. ⇑ Corresponding author. E-mail addresses: [email protected] (M.J.T.N. Timmermans), Many of these poorly supported groupings are found in the same [email protected] (D.C. Lees), [email protected] (T.J. Simonsen). parts of the Lepidoptera Tree of Life where morphology alone has http://dx.doi.org/10.1016/j.ympev.2014.05.031 1055-7903/Ó 2014 Elsevier Inc. All rights reserved. 170 M.J.T.N. Timmermans et al. / Molecular Phylogenetics and Evolution 79 (2014) 169–178 failed to yield conclusive evidence for evolutionary relationships. It representatives of 22 Lepidoptera superfamilies (Table 1). PCR is therefore clear that despite considerable efforts, we still remain reactions were performed with Takara LA taq and followed cycling far from a unified consensus of the phylogeny of the higher conditions described in Timmermans et al. (2010). Two primer Lepidoptera. For example, sister groups of some of the most prom- pairs were used for long-range PCR: One pair (SytbF/reverse inent lepidopteran radiations (Papilionoidea and Gelechioidea) complement of Jerry, JerryRC: Simon et al., 1994) targeting a 7 kb remain as yet unknown. To maximise our chances for arriving at fragment running from COI to CytB across the AT-rich region, such a ‘‘consensus hypothesis’’, we need to look at broadening both and one pair (Jerry: Simon et al., 1994/SytbR) targeting the oppo- taxon and character sampling in future phylogenetic studies. site 9 kb half of the circular genome (Fig. 1). Not all PCRs were Within the realm of molecular characters, full mitochondrial successful, and for some species one fragment was obtained genomes can be a data-rich and relatively accessible source of (Table 1; Fig. 2). For Pennisetia hylaeiformis the JerryRC-SytbR PCR information, and promising results have been obtained for other failed and a shorter fragment was targeted by replacing the JerryRC insect families and orders (e.g. Timmermans et al., 2010; Haran primer with stevCO3F (GCWTGATAYTGACAYTTYGT) located in et al., 2013; Simon and Hadrys, 2013). So far, the mt-phylogenomic COIII. In contrast to Timmermans et al. (2010), who used Roche’s studies with the most comprehensive lepidopteran taxon sampling 454 platform, sequencing was performed using Illumina’s Solexa remain the Kim et al. (2011), Lu et al. (2013) analyses that focused technology. Fragments were pooled before TruSeq library almost entirely on the polyphyletic group ‘‘Macrolepidoptera’’ construction (800 bp insert size) and paired-end sequenced (PE: (essentially Papilionoidea plus Macroheterocera sensu van 2 Ã 250 bp; V2 Reagent Kit) on an Illumina Miseq aiming for 1% Nieukerken et al., 2011). of the machines run capacity. In addition to these Lepidoptera Here we characterise full mitochondrial genomes using a samples, the pool also contained 22 partial beetle mitochondrial method that relies on the in silico separation of DNA sequence genomes, which are not further discussed here. Three ‘baits’ were data from pooled PCR fragments (Timmermans et al., 2010). We generated to assign anonymous contigs to the voucher specimens. acquired mitochondrial genomes of a diverse set of lepidopterans These baits included a 400 bp fragment of CytB (primer pair: and present the most comprehensive mt-phylogenomic study of SytbF + SytbR) and two COI fragments (primer pairs LepCoxF1: Lepidoptera to date (mainly focused on the subgroup Heterone- ACNAATCATAARGATATTGGNAC + JerryRC and Jerry + sCOIR: CARA- ura). We explore the evolutionary patterns of the different genes TTTCDGARCATTG) (Fig. 1). ‘Bait’ sequences were bi-directionally in the mt-genome across heteroneuran Lepidoptera and assess sequenced using Sanger technology and edited in Geneious version the utility of mt-genomes in lepidopteran phylogenetics. 5.6 (Biomatters, Auckland, New Zealand). 2. Materials and methods 2.2. Sequence assembly and annotation 2.1. PCR amplification and sequencing Raw sequence reads were pre-processed using the Trimmomat- ic (Lohse et al., 2012) and Prinseq-lite (Schmieder and Edwards, Wet-lab methods are based on those described in Timmermans 2011) perl scripts. Bases with a Phred quality score below 20 were et al. (2010). Mitochondrial genomes were PCR amplified from trimmed. Sequence reads with a high quality length below 150 bp Table 1 Details of Lepidoptera samples (29) subjected to mitochondrial genome sequencing. Superfamily Family Genus Species Voucher number Sequence length (bp) Mean coverage Accession number 1À Papilionoidea Papilionidae Papilio dardanus BMNH746800 15,337 405 JX313686 2 Geometroidea Geometridae Chloroclysta truncata BMNH1043250 15,828 428 KJ508061 3 Drepanoidea Drepanidae Drepana arcuata CWM-96-0579* 15,300 254 KJ508053 4 Incertae sedis Doidae Doa BOLD:AAC3419 05-srnp-63413* 15,228 185 KJ508058 5 Noctuoidea Noctuidae Acronicta