First complete mitochondrial genome of a Gripopterygid stonefly from the sub-order Antarctoperlaria: Zelandoperla fenestrata

Abstract

Plecoptera (stoneflies) are an anciently derived order of freshwater insects with two divergent suborders Antarctoperlaria in the southern hemisphere and Arctoperlaria primarily in the northern hemisphere. In this paper, we present the first published mitochondrial genome of an Antarctoperlarian stonefly: the New Zealand stonefly Zelandoperla fenestrata. The Z. fenestrata mitochondrial genome is 16,385 bp, with the typical insect mitogenome complement of 13 protein-coding genes, 22 tRNAs, and two rRNA genes, along with a A + T rich control region. This mitogenome will be of interest to the study of evolutionary divergence within Polyneoptera, and will aid in phylogeographic studies of this species.

Keywords:

• Plecoptera
• polyneoptera
• mitogenome

Introduction

Stoneflies (order: Plecoptera) are a widespread order of winged freshwater insects, with fossils dating back to the early Permian (Sinichenkova 1997; Zwick 2000; Bethoux et al. 2011; Cui et al. 2016). Plecoptera is divided into two sub-orders: Antarctoperlaria in the southern hemisphere and Arctoperlaria, primarily in the northern hemisphere. These two sub-orders diverged around 121 Ma (McCulloch et al. 2016). Currently, there are twenty-three published mitochondrial genomes of stoneflies, all from Arctoperlaria.

The stonefly Zelandoperla fenestrata species group (Z. fenestrata, Z. pennulata, Z. tillyardi) is widely distributed across New Zealand (McLellan 1977, 1999). Because of significant population genetic structure, and the presence of multiple non-dispersive flightless forms on multiple mountain ranges, understanding the phylogeography and evolutionary history of this species is of great interest (McCulloch et al. 2009; Dussex et al. 2016; Veale et al. 2018).

Methods

Library preparation and Illumina sequencing

A single Z. fenestrata nymph identified based on the key in (McLellan 1999) was collected from an upper tributary of the Logan Burn stream in Central Otago, New Zealand (45.46884S, 169.96146E) elevation 1007 m, and frozen from live at −70 °C until DNA extraction. We then dissected out the head and forelegs and used a Qiagen DNeasy blood and tissue kit to extract the DNA. We then created a 350 bp paired-end genomic library using an Illumina TruSeq Nano DNA Neoprep, with an average insert size of 607 bp. This was sequenced using an Illumina Hi-Seq with 125 bp reads. DNA remains stored at the University of Otago Zoology Department.

Sequence assembly and analysis

We used CUTADAPT (Martin 2011) to remove adapter sequences and trim sequences (cut off =30, minimum length =50). We then used MITOBIM v1.8 (Hahn et al. 2013) to extract and align mitochondrial sequences using a COI barcode sequence as a seed (Genbank Accession GQ414619.1). The mitogenome sequence was annotated using the MITOS web server (Bernt et al. 2013). We aligned the sequence with all published Plecoptera mitochondrial genomes, along with the mayfly Ephemera orientalis as an outgroup in Geneious® 9.1.8. (Kearse et al. 2012) then created a Baysian phylogenetic tree using MrBayes (2,500,000 chain length, 250 sampling interval, 10% burn-in, using an HKY + G + I mutational model (as determined in Topali2 Milne et al. 2009).

Results

We obtained a mitochondrial genome of 16,385 bp, (average coverage 98×) Genbank Accession KY522907. The Z. fenestrata mitogenome retains the typical insect mitogenome gene set, including 13 protein-coding genes (ND1-6, COX1-3, ND4L, ATP8, ATP6, and CYTB), 22 tRNA genes, two ribosomal RNAs and a control region. The phylogenetic tree reinforces the believed deep divergence between Antarctoperlaria and Arctoperlaria (Figure 1).

Figure 1. Bayesian mitogenome phylogenetic tree showing the divergence of Zelandoperla fenestrata (order: Antarctoperlaria) to all other published stonefly mitogenomes (all order: Arctoperlaria). Stonefly family listed to the right.

Discussion

This mitogenome confirms the placement of Antarctoperlaria as basal and divergent from Arctoperlaria (Chen and Du 2018) – which had previously been suggested both from morphology (Zwick 2000) and from COI sequences (McCulloch et al. 2016). As this is the first mitogenome from any member of Antarctoperlaria, this sequence should be of interest for phylogenetic reconstructions within Polyneoptera, and for looking at mitochondrial evolutionary rates within Plecoptera.

Acknowledgements

The authors wish to thank Aaron Jeffs, NZGL and Darren Hart for their contributions in helping to analyze and sequence the genome.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was funded by Marsden fund grant (UOO1412).