EUROPEAN JOURNAL OF ENTOMOLOGYENTOMOLOGY ISSN (online): 1802-8829 Eur. J. Entomol. 114: 407–415, 2017 http://www.eje.cz doi: 10.14411/eje.2017.052 ORIGINAL ARTICLE Tandem duplication of two tRNA genes in the mitochondrial genom e of Tagiades vajuna (Lepidoptera: Hesperiidae) FANG-FANG LIU 1, *, YI -PING LI 1, 2, *, IVAN JA KOVLIĆ 3 and XIANG-QUN YU AN 1, ** 1 Ke y Laboratory of Plant Protection Resources and Pest Management, Ministry of Education; College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; e-mails: [email protected] (F.-F.L.), ly [email protected] (Y.-P.L), yua [email protected] (X.-Q.Y.) 2 Key Laboratory of Applied Entomology, Northwest A&F University, Yangling, Shaanxi 712100, China 3 De partment of Fisheries, Beekeeping, Game Management and Special Zoology, Fa culty of Agriculture, University of Zagreb, Svetošimunska cesta 25, 10000 Za greb, Croatia; e-mail: i [email protected] Key words. Lepidoptera, Hesperiidae, Pyrginae, Eudaminae, Tag iades vajuna, mit ochondrial phylogenomics, tRNA duplication, butterfl ies, skippers Abstract. To explore the debated phylogenetic relationship of two Hesperiidae subfamilies, Pyrginae and Eudaminae, and con- tribute to the understanding of the evolution of mitogenomic architecture in butterfl ies, we sequenced the complete mitogenome of Tag iades vajuna. The mitogenome is a typical circular duplex molecule of 15,359 bp. Apart from the standard 22 tRNAs, it has a tandem duplication of trnS(AGN) and trnE, which is unique in lepidopteran insects. Comparison with Ctenoptilum vasava indi- cates that the trnS1 duplication is not an ancestral state shared with other species of Tagiadini. Independent origin of the trnS1 duplications was further confi rmed by the reconstruction of the ancestral character state based on the topology of the phylogram. Furthermore, comparative analysis of mitogenomes with and without tRNA duplications indicates that tRNA duplication does not alter the codon usage pattern. The mitogenome has negative AT- and GC-skews, and it is highly A+T-biased (79.7%). The AT-rich (or control) region (283 bp) contains “ATAGA” and “ATTTA” motifs. Regardi ng the phylogenetic analysis, we found that removal of the third codon position (3CP) from datasets used for the mitochondrial phylogenomics of Hesperiidae is likely to produce results that are more consistent: Pyrginae were rendered paraphyletic by Eudaminae in both analyses of the dataset from which the 3CP was removed (13 PCGs + all RNAs), but inclusion of the 3CP resulte d in a destabilized topology, resulting in both monophyly and polyphyly. We conc lude that even shallow-phylogenies of insects should pay close attention to compositional and mutational biases in mitogenomes. INTRODUCTION itance and general absence of recombination, they are often Skippers (Hesperiidae) are a species-rich family (ap- the tool of choice for resolving phylogenetic relationships proximately 3,587 species), which makes up one fi fth of among insects (Brown & Wilson, 1979; Ballard & Whit- the butterfl y species in the world. Previous studies have lock, 2004; Gissi et al., 2008; Nelson et al., 2012; Simon found that neither three mitochondrial genes (Yuan et al., & Hadrys, 2013; Wan et al., 2013; Cameron, 2014; Dai et 2015), nor ten nuclear and mitochondrial markers (Sahoo al., 2016). There are some limitations to the application of et al., 2016), were capable of providing suffi cient phylo- mitochondrial phylogenomics in insects, such as composi- genetic resolution to clarify the evolutionary relationships tional heterogeneity (Cameron, 2014) and mutation satura- among the Hesperiidae. Particularly diffi cult to resolve are tion in the third codon position (3CP) of protein-coding the relationships among the subfamilies Pyrginae, Eudami- genes (PCGs) (Cameron et al., 2012). However, these limi- nae and Euschemoninae. This led the above authors to pro- tations are relatively well-understood in deep-phylogenies pose that future attempts would need much larger datasets. of Insecta lineages (Cameron et al., 2007; Sheffi eld et al., Due to several advantageous characteristics of mito- 2009; Cameron, 2014; Song et al., 2016), the extent of their chondrial genomes, which include small size, abundance effects remains incompletely investigated (and occasion- in tissues, strict orthology of encoded genes, presence of ally even ignored, see below) in shallow-phylogenies of genes/regions evolving at different rates, uniparental inher- insects. This might be a consequence of an insuffi cient * Contributed equally. ** Corresponding author; e-mail: yua [email protected] Final formatted article © Institute of Entomology, Biology Centre, Czech Academy of Sciences, České Budějovice. An Open Access article distributed under the Creative Commons (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). 407 Liu et al., Eur. J. Entomol. 114: 407–415, 2017 doi: 10.14411/eje.2017.052 number of mitogenomes being available for such phylog- and investigate tRNA gene duplication in the broad phylo- enies. The latest two studies of the family Hesperiidae, genetic context of the family Hesperiidae. employing the mitochondrial phylogenomics approach (Zhang et al., 2017b, c), found that the subfamilies Pyrgi- MATERIALS AND METHODS nae and Eudaminae exhibit unresolved polytomies. As we Sample collection and DNA extraction suspect that their results may have been infl uenced by the A single specimen of an adult T. vajuna (Maruyama, 1991) aforementioned limitations of this approach, we adopt the was captured by Shi-Hong Jiang on 14th November 2015 in Yuan- most commonly used strategy to alleviate compositional shan Park, Shenzhen City, Guangdong Province, China (22°43´– heterogeneity: removal of the 3CP of PCGs. In addition, to 23°15´N; 114°14´–115°16´E). The specimen was taxonomically improve taxon sampling and phylogenetic resolution, we identifi ed by its morphological characteristics (particularly the sequenced the mitochondrial genome of a species belong- genitalia) (Yuan et al., 2015), and cox1 barcoding (Fig. S1) using 491 of the 493 sequences available for Pyrginae in the BOLD ing to the subfamily Pyrginae, Tagiades vajuna. To assess database (Ratnasingham & Hebert, 2013) (two incomplete Py r- the effects of this strategy, we also conducted analyses on ginae barcodes were excluded). The complete specimen was a dataset comprising all three codon positions of all PCGs. immediately preserved in 100% ethanol and stored at –20°C in Although the removal of RNAs (rRNAs and tRNAs) has the Entomological Museum of the Northwest A&F University, been a common approach used in studies utilizing mitog- Yangling, Shaanxi Province, China. The total DNA was extracted enomic data to investigate the phylogenetic relationships from the thoracic muscles following the manufacturer’s instruc- among the major lineages of Lepidoptera (Kim et al., 2011; tions (EasyPureR Genomic DNA Kit, TRAN, TransGen, Beijing, Yang et al., 2015), there is no evidence of signifi cant in- China). As T . vajuna is an unprotected invertebrate species, no congruence in the phylogenetic signals between RNAs and permits were required for this study. PCGs in insects (Cameron et al., 2007; Cameron, 2014). Sequence analysis RNAs can actually carry a considerable phylogenetic sig- The complete mitochondrial genome of T. vajuna was se- nal and their inclusion has had positive effects on nodal quenced on an Illumina HiSeq2000 system by the Genesky Bio- support for some lineages within Lepidoptera (Wan et al., technologies Inc. company (Shanghai, China). Illumina sequenc- 2013). Thus we have adopted a strategy of maximizing the ing data were quality-trimmed with Trimmomatic v0.35 (Bolger amount of data by including all 37 mitogenomic genes: 13 et al., 2014) and used for mitochondrial DNA genome assembly PCGs (excluded/included 3CP), two rRNAs and 22 tRNAs. via a two-step MIRA4/Mitobim combined pipeline (Hahn et al., 2013), which implements a hybrid mapping and assembly ap- Beyond mitochondrial phylogenomics, mitogenomic proach for the targeted assembly of homologous sequences. The gene duplication and the existence of pseudogenes is of annotation of the mitochondrial DNA sequence was carried out interest in studies of the evolutionary history and mecha- in Geneious 8.1.3 (Biomatters, Auckland, New Zealand), using nisms of gene rearrangement and recruitment (Ye et al., the mitogenome of another species of Hesperiidae, Parnara gut- 2016). Although tRNAs are considered to be the most tata (Hesperiidae: Hesperiinae; GenBank: NC_029136) (Shao “expendable” among the genes encoded by the mitochon- et al., 2015), as a reference. Protein-coding genes (PCGs) were drial genomes and their content and order in mitogenomes determined by fi nding the ORFs (employing codon table 5), and is rather variable (Gissi et al., 2008), only four tRNA du- rRNAs (12S and 16S) were identifi ed using the MITOS Web plications and/or tRNA pseudogenes are recorded in the Server (Bernt et al., 2013). Transfer RNAs, including the two duplicated tRNAs, trnE and trnS(AGN), were also identifi ed by superfamily Papilionoidea. Among these four, one species MITOS and by manually inspecting the potential cloverleaf sec- belongs to Lycaenidae, Coreana raphaelis (trnS1 duplica- ondary structures and anticodons. Finally, all genes were visually tion) (Kim et al., 2006), one to Nymphalidae, Acraea isso- inspected against the reference mitogenome via alignments in ria (trnI pseudogene) (Hu et al., 2010) and the remaining Geneious. Nucleotide