Gene 547 (2014) 136–144 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene The complete mitogenome of Apocheima cinerarius (Lepidoptera: Geometridae: Ennominae) and comparison with that of other lepidopteran insects Shuxian Liu a,b,DayongXuea, Rui Cheng a,b, Hongxiang Han a,⁎ a Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China b University of the Chinese Academy of Sciences, Beijing 100049, China article info abstract Article history: The complete mitochondrial genome (mitogenome) of a female flightless geometrid moth Apocheima cinerarius Received 19 February 2014 was found to be 15,722 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 Received in revised form 22 May 2014 ribosomal RNA (rRNA) genes, and a control region. The A + T content of the complete mitogenome is 80.83%. The Accepted 21 June 2014 AT skew value ([A − T] / [A + T]) is 0.027. The 13 PCGs of the mitogenome start with typical ATN codons, except Available online 24 June 2014 for cox1 with the start codon CGA. All the tRNA genes have typical cloverleaf secondary structures, except for Keywords: trnSer(AGN). The secondary structures of rrnL and rrnS were predicted. Six structural domains including con- fi Mitochondrial genome served regions (IV, V) and variable regions (I, II, III, VI) were identi ed in the secondary structure of rrnL. The sec- Apocheima cinerarius ondary structure of rrnS consists of 3 structural domains. The control region of A. cinerarius begins with conserved Secondary structure motifs of “ATAGA” + 19-bp poly T. It also contains a microsatellite-like (TA)26, a stem-and-loop structure, and a Phylogeny poly-A stretch. Phylogenetic analysis showed that Geometroidea is more closely related to Bombycoidea than to Noctuoidea. A. cinerarius is more closely related to Biston panterinaria than to Phthonandria atrilineata, which is in accordance with the conventional morphology-based classification. © 2014 Elsevier B.V. All rights reserved. 1. Introduction also contains an A + T-rich region of variable length, which is the largest noncoding area, functioning in the regulation of transcription and Apocheima cinerarius (Erschoff, 1874) (Lepidoptera: Geometridae: replication (Wolstenholme, 1992). Geometridae is the third largest Ennominae) is widely distributed in northern China, southeast Russia, lepidopteran family, with more than 26,000 described species, but and Central Asia. The moth, one of the female flightless moths in only 2 Geometridae (Phthonandria atrilineata and Biston panterinaria) Geometridae, is univoltine. It overwinters by pupa and emerges from mitogenomes have been published (Yang et al., 2009, 2013). To date, early March to early May. A. cinerarius outbreaks occur frequently in there has been no report of the complete mitogenome of a female flight- northern China. The moth can defoliate deciduous trees in a few days, less moth. damaging the forest landscape and destroying the regional ecological In the present study, the complete mitogenome of A. cinerarius was balance (Chu, 1981). A. cinerarius has the most economic significance sequenced. The genomic organization, base composition, PCGs, tRNA among the described female flightless moths of Geometridae. Research genes and rRNA structures, and A + T-rich region were analyzed, with on A. cinerarius has mostly focused on its biology and control methods a focus on the A + T-rich region and the secondary structure for rrnL (Zhang et al., 2011); there have been no reports of its mitogenome. and rrnS. The phylogenetic relationships among lepidopteran super- The insect mitogenome DNA is a circular molecule 14–16 kbp in families were reconstructed using the nucleotide sequence of 13 PCGs length. It contains 13 protein-coding genes (PCGs), 2 ribosomal RNA for 52 lepidopteran species. The relationships among A. cinerarius, genes (large and small rRNAs), and 22 transfer RNA (tRNA) genes. It P. atrilineata,andB. panterinaria were comparatively analyzed. Abbreviations: Mitogenome, mitochondrial genome; PCGs, protein coding genes; atp6 2. Material and methods and atp8, atp synthase subunit 6 and 8 genes; cox1-3, cytochrome c oxidase 1–3 genes; cytb, cytochrome b gene; nad1-6 and nad4L, NADH dehydrogenase subunit 1–6 and 4L 2.1. DNA extraction genes; rRNA, ribosomal RNA; rrnL and rrnS, large and small subunit ribosomal RNA; tRNA, transfer RNA; PCR, polymerase chain reaction; AIC, Akaike information criterion. An adult specimen of A. cinerarius was collected from Lulong, Hebei, ⁎ Corresponding author. ′ ′ E-mail addresses: [email protected] (S. Liu), [email protected] (D. Xue), China (118°52 ,39°52) in March 2007. The specimen was preserved in [email protected] (H. Han). anhydrous ethanol at −20 °C and stored in the Lepidoptera systematics http://dx.doi.org/10.1016/j.gene.2014.06.044 0378-1119/© 2014 Elsevier B.V. All rights reserved. S. Liu et al. / Gene 547 (2014) 136–144 137 group of the Institute of Zoology, Chinese Academy of Sciences. DNA 2.4. Phylogenetic analysis was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Beijing, China). The phylogenetic relationships of Lepidoptera were reconstructed using a concatenated set of 13 PCGs of 52 complete mitogenomes 2.2. PCR amplification and sequencing (51 obtained from GenBank, A. cinerarius from the present study) (Table. 2). Thitarodes yunnanensis and Thitarodes renzhiensis (Cao Short fragments of the A. cinerarius mitogenome were PCR-amplified et al., 2012) of Hepialoidea in Exoporia were used as outgroups. The with previously published conserved primers (Clary and Wolstenholme, 13 PCG nucleotide sequences were aligned and translated into amino 1985; Hebert et al., 2004; Kim et al., 2012; Sezonlin et al., 2006; Simon acid sequences on the basis of the invertebrate mitochondrial genetic et al., 1994). Specific primers were designed from the short fragments code with deleted stop codons, using MEGA 5.0. The GTR + I + G using Primer Premier 5 (Premier Biosoft International) (Table 1). PCR model was selected from 88 models by the Akaike information criterion reactions for long fragments were performed in a 25-μl volume with (AIC) with the jModelTest 0.1 (Posada, 2008). The concatenated sets of 0.625 U of LA Taq (Takara, China), 1 μlofDNA,2.5μl10×LATaqbuffer nucleotide sequences of the 13 PCGs were used to reconstruct the (plus Mg2+), 4 μl dNTPs, and 16 pmol of each primer. PCR reactions for phylogenetic relationships by Bayesian inference and Maximum short fragments (b1 kb) were performed using the following procedure: likelihood (ML) methods. The Bayesian analysis was performed 94 °C for 2 min; 5 cycles of 40 s at 94 °C, 40 s at 45 °C, and 1.5 min at using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003). The 72 °C; and 35 cycles of 40 s at 94 °C, 40 s at 51 °C, and 1.5 min at 72 °C, MCMC analysis was run for 10,000,000 generations, following a followed by 72 °C for 10 min (Hebert et al., 2004). The long segments burn-in series of 1000. Maximum likelihood (ML) analysis was con- (3–5 kb) were amplified with specific primers using the following ducted using the program of RAxMLv7.0.4 (Stamatakis, 2006)with procedure: 94 °C for 2 min, 35 cycles of 30 s at 94 °C, 30 s at 45–55 °C, conducting 1000 bootstraps. The GTRGAMMA model was used for and 12 min at 68 °C, followed by 68 °C for 10 min. The PCR products all 13PCGs and partitions. were detected by 1% agarose gel electrophoresis and directly sequenced with ABI PRISM 3730xl capillary sequencers. 3. Results 2.3. Sequence assembly, gene annotation, and secondary structure prediction 3.1. Genome organization and base composition The sequences were run using the NCBI BLAST program to determine sequencehomology.Thecompletemitogenomewasassembledfromthe The complete mitogenome of A. cinerarius was found to be 15,722 bp overlapping short sequences with ChromasPro (www.technelysium.com. in length, containing 13 PCGs, 22 tRNA genes, 2 rRNA genes, and 1 con- au/ChromasPro.html), and open reading frames (ORFs) were identified trol region (Fig. 1), like other lepidopteran genomes. The mitogenome using the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) sequence has been deposited in GenBank under accession number at NCBI. The nucleotide sequences were aligned with the known KF836545. Twenty-three genes are transcribed on the J strand and the mitogenome sequences of closely related species using ClustalX 1.83 remaining 14 are transcribed on the N strand. The nucleotide composi- (Thompson et al., 1997). The base composition was calculated using tion is (A) 41.51%, (C) 11.37%, (G) 7.8%, and (T) 39.32%. The AT nucleo- BioEdit, and codon usage was calculated using MEGA 5.0 (Tamura tide content is 80.83%. The AT skew value ([A − T] / [A + T]) (Perna and et al., 2011). The 22 tRNA cloverleaf secondary structures and anticodon Kocher, 1995b; Wei et al., 2010) is 0.027. sequences were determined with tRNAscan-SE version 1.21 (lowelab. ucsc.edu/tRNAscan-SE/)(Lowe and Eddy, 1997) and alignment with the known mitogenome sequences of closely related species. The rrnL 3.2. Protein-coding genes and rrnS secondary structures were inferred from Mfold Web Server (http://mfold.rna.albany.edu/?q=mfold) and comparison with rRNA The mitogenome of A. cinerarius contains 13 PCGs starting with the secondary structures proposed for other insects, including Grapholita typical ATN codons, except for cox1 with the CGA start codon. There molesta (Lepidoptera: Tortricidae), Manduca sexta (Lepidoptera: are 3766 codons excluding the start and termination codons. UUA Sphingidae), Phalera flavescens (Lepidoptera: Notodontidae), and (Leu), UUU (Phe), and AUU (Ile) are the most abundant amino acid co- Ochrogaster lunifer (Lepidoptera: Notodontidae) (Cameron and dons. Codon usage in PCGs is biased, using more A and T than G and C.