The complete mitochondrial genome of the emperor dragonfly Anax imperator LEACH, 1815 (Odonata : Aeshnidae) via NGS sequencing

Abstract

Here we report the complete mitochondrial genome of the emperor dragonfly, Anax imperator (Odonata: Aeshnidae) as the first of its genus. Data were generated via next generation sequencing (NGS) and assembled using an iterative approach. The typical metazoan set of 37 genes (13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes) was detected in the same gene order as in other odonate mitogenomes. However, only three intergenic spacer regions are present in A. imperator lacking the distinct s5 spacer, which was regarded as informative feature of the odonate suborder Anisoptera (dragonflies) but absent in Zygoptera (damselflies). With 16,087 bp, it is the longest anisopteran mitogenome to date, mainly due to the long A + T-rich control region of 1291 bp.

Keywords:

• Mitochondrial genome
• odonata
• Anax imperator
• aeshnidae
• anisoptera
• s5 intergenic spacer

The emperor dragonfly, Anax imperator, is a widespread and common species in the old world inhabiting all types of standing- and slow-running freshwater ecosystems. It was one of the first odonate species for which a recent range shift northwards (e.g. Parr 2010) and towards higher altitudes (Westermann 2003; Hunger et al. 2006) was noticed due to global climate change. The first records of this species in Sweden were 2002 (Ott 2010). In only 11 years A. imperator crossed a distance of 970 km northwards through Scandinavia (Nielsen 1998; Lejfelt-Sahlén 2007). The larvae of this large dragonfly species are known to be very aggressive (e.g. Beutler 1985) and will invade and significantly influence the native species composition of freshwater ecosystems. Genetic and comparative genomic studies on range shift, expansion, and adaptive potential of this species are of great interest to further elucidate the impact of global change on flying insects. To date for A. imperator, a panel of 10 nuclear microsatellite loci and partial mitochondrial genes (cox1, nad1, and both rRNAs) were established so far to serve in various phylogenetic studies (Misof et al. 2001; Hadrys et al. 2007; Fleck et al. 2008; Rach et al. 2008; Bergmann et al. 2013). To consequently proceed towards a comparative genomic approach one first step is the unravelling and comparison of mitogenomes, e.g. their gene content, arrangements, and genealogical relationships.

As for the A. imperator mitogenome, a standard phenol–chloroform protocol by Hadrys et al. (1992) was used to extract total genomic DNA from flight muscles of a single individual collected in Southern France (43°36′17.7″N 4°48′34.4″E). DNA was submitted for library preparation and whole genome sequencing on an llumina HiSeq2000 (75 bp paired-end reads) to the Yale Center for Genome Analyses (YCGA, http://www.ycga.yale.edu). Different mitochondrial gene sequences containing partial nad1, cox1, 12S rRNA, and 16S rRNA genes (accession numbers: KC912228.1, KF584974.1, EU477652.1 and EU183256.1) were used as reference seeds for a subsequent assembly employing Genious v.8.1.5 (http://www.geneious.com/). For mitochondrial genome annotation, the MITOS WebServer (mitos.bioinf.uni-leipzig.de/index.py) was applied and results were checked manually using BLAST (Altschul et al. 1990) and available odonate mitochondrial genomes (e.g. Yu et al. 2014; Chen et al. 2015). Transfer RNA genes were predicted using both, the tRNAscan-SE v.1.21 Search Server (Lowe & Eddy 1997) and ARWEN v.1.2 (Laslett & Canbäck 2008).

The complete circular mitochondrial genome sequence of A. imperator (GenBank accession number #KX161814) with the length of 16,087 bp is the largest known mitogenome among Anisoptera. It exhibits the standard metazoan gene content of 37 genes, comprising 13 protein-coding genes, 22 tRNA genes, and two rRNA genes which are identically arranged as in the few other odonate mitochondrial genomes (e.g. Simon & Hadrys 2013; Lorenzo-Carballa et al. 2014; Chen et al. 2015; Yu et al. 2014; Feindt et al. 2016). Overall base frequency is 76.0% AT-biased, for the 1291 bp long control (A + T rich) region even 93.5%. All standard mitochondrial invertebrate start codons are found, in detail ATT (nad5), ATA (nad2, nad3), TTG (cox1, nad1), ATC (atp8, nad6), and ATG (cox2, atp6, cox3, nad4, nad4l, cob). Two proteins (cox2, nad5) possess a single T as an incomplete stop codon, requiring post-transcriptional polyadenylation whereas all others protein-coding genes use TAA as stop codon (Table 1). The gene length of tRNA genes ranges from 65 bp to 73 bp and all tRNAs can be folded in the typical cloverleaf structure, except the D-replacement tRNA trnS1. Further, two pseudo-tRNA genes were detected by the tRNA prediction software ARWEN v.1.2 (Laslett & Canbäck, 2008) which were both D-Loop tRNAs and located inside the cox2 sequence and in trnA/trnR, respectively. Therefore, their functionality remains questionable.

Table 1. Mitochondrial genome organization and gene content of A. imperator with detailed description of gene boundaries, strand, gene length (in bp) as well as start and stop codons for protein-coding genes and anticodons for tRNA genes, respectively.

Gene/region	Strand	Start position	Stop position	Length (bp)	Anti/start codon	Stop codon
trnI	+	214	281	68	GAT	/
trnQ	−	278	347	70	TTG	/
trnM	+	352	420	69	CAT	/
nad2	+	424	1419	996	ATA	TAA
trnW	+	1417	1487	71	TCA	/
trnC	−	1479	1543	65	GCA	/
trnY	−	1545	1613	69	CTA	/
s1	NA	1614	1653	40	/	/
cox1	+	1654	3192	1539	TTG	TAA
trnL2	+	3187	3256	70	TAA	/
cox2	+	3256	3943	688	ATG	T(aa)
trnK	+	3944	4016	73	CTT	/
trnD	+	4016	4084	69	GTC	/
atp8	+	4084	4245	162	ATC	TAA
atp6	+	4239	4916	678	ATG	TAA
cox3	+	4916	5704	789	ATG	TAA
trnG	+	5704	5768	65	TCC	/
nad3	+	5,766	6,122	357	ATA	TAA
trnA	+	6122	6190	69	TGC	/
trnR	+	6189	6,258	70	TCG	/
trnN	+	6258	6324	67	GTT	/
trnS1	+	6325	6392	68	GCT	/
trnE	+	6392	6460	69	TTC	/
trnF	−	6459	6526	68	GAA	/
nad5	−	6525	8254	1730	ATT	T(aa)
trnH	−	8255	8322	68	GTG	/
nad4	−	8322	9665	1344	ATG	TAA
nad4l	−	9659	9952	294	ATG	TAA
trnT	+	9954	10,022	69	TGT	/
s2	NA	10,023	10,045	23	/	/
trnP	−	10,046	10,111	66	TGG	/
nad6	+	10,113	10,634	522	ATC	TAA
cob	+	10,634	11,767	1134	ATG	TAA
trnS2	+	11,766	11,832	67	TGA	/
s3	NA	11,833	11,849	17	/	/
nad1	−	11,850	12,800	951	TTG	TAA
trnL1	−	12,801	12,868	68	TAG	/
l-rRNA	−	12,810	14,180	1371	/	/
trnV	−	14,167	14,236	70	TAC	/
s-rRNA	−	14,239	15,008	770	/	/
A + T-rich (control) region	NA	15,009	212	1291	/	/

Transfer RNAs are given in the one-letter amino acid code with the corresponding anticodons. Intergenic spacer regions are numbered (s1–s3).

However, in contrast to the known other anisopteran mitogenomes, only three intergenic spacer regions were discovered (see Table 1). These are located between trnY/cox1, trnT/trnP, and trnS2/nad1. They are also present in other odonates (Anisoptera and Zygoptera), e.g. Ischnura elegans (Feindt et al. 2016), Ischnura pumilio (Lorenzo-Carballa et al. 2014), Megaloprepus caerulatus (Feindt et al. 2016), or Brachythemis contaminata (Yu et al. 2014). The latter, an anisopteran species additionally shows a fourth spacer region between nad1/trnL2 that is asserted to be typical for Anisopterans and lacking in Zygopterans (Lin et al. 2010). This spacer, commonly called s5 (though counting and numbering spacer regions is not consistent between most mitogenome publications) is not present in Anax. Consequently, the absence of this spacer refutes the theory of being a putative distinctive feature between Anisoptera and Zygoptera and stresses the necessity to analyze more mitogenomes within Odonata to allow stronger, reliable assumptions about phylogenetically informative mtDNA characteristics. The phylogenetic position of A. imperator in the context of all available anisopteran mitogenomes to date (3 May 2016) is displayed in Figure 1 and so far consistent with other gene tree phylogenies.

Figure 1. Neighbour-Joining Tree of A. imperator within all available anisopteran odonate species (03 May 2016): Orthetrum triangulare (AB126005.1), Hydrobasileus croceus (NC_025758.1), B. contaminata (NC_026305.1), Ictinogomphus sp. (KM244673) and Davidius lunatus (NC_012644.1). The phylogeny was reconstructed based on 13 mitochondrial protein-coding genes via Paup with 1000 bootsrap replicates and Euphea formosa (NC_014493.1) as an outgroup.

Disclosure statement

The authors declare no conflict of interest to other working groups.