The complete mitochondrial genome of the Madagascar mother-of-pearl butterfly Salamis anteva (Insecta: Lepidoptera: Nymphalidae)

Abstract

The Madagascar mother-of-pearl (Salamis anteva) is a leaf-mimicking butterfly endemic to forests in southern Madagascar. Genome skimming by Illumina sequencing permitted assembly of a complete S. anteva circular mitogenome of 15,201 bp consisting of 80.6% AT nucleotides and with an arrangement of 22 tRNAs, 13 protein-coding genes, two rRNAs and a control region common to all butterflies. The mitogenome includes a COX1 gene with an atypical CGA start codon and four genes exhibiting incomplete stop codons. Phylogenetic reconstruction places S. antvea as sister to Yoma sabina within a monophyletic nymphalid tribe Junoniini. This is consistent with previous molecular phylogenetic hypotheses.

Keywords:

• Illumina sequencing
• mitogenomics
• Lepidoptera
• Nymphalidae
• Nymphalinae

The Madagascar mother-of-pearl butterfly, Salamis anteva (Lepidoptera: Nymphalidae), is a leaf-mimicing endemic species (Suzuki et al. 2014) that inhabits forested regions in the southern portions of Madagascar (Lees et al. 2003). It has been suggested as an indicator species for the health of these forested habitats (Kremen 1994). Here, we report the complete mitochondrial genome sequence of S. anteva from a specimen (ID: Sant2015.1) collected in Andasibe, Madagascar in March 2015 that has been pinned, spread, and deposited in the Wallis Roughley Museum of Entomology, University of Manitoba (voucher JBWM0379998).

DNA was prepared (McCullagh and Marcus 2015) and sequenced by Illumina MiSeq (San Diego, California) (Peters and Marcus 2017). The mitogenome of S. anteva (Genbank MH917707) was assembled by Geneious 10.1.2 from 7,876,900 paired 300 bp reads using a Junonia lemonias (Nymphalidae, KP941756) (McCullagh and Marcus 2015) reference mitogenome and annotated by comparison with J. lemonias and Precis andremiaja (Nymphalidae: MH917706) (Lalonde and Marcus 2018) mitogenomes. The S. anteva nuclear rRNA repeat (Genbank MH917709) was assembled and annotated using Meroptera pravella (MF073208) (Living Prairie Mitogenomics Consortium 2017), Samia cynthia ricini (Saturniidae, AF463459) (Wang et al. 2003) and Papilio xuthus (Papilionidae, AB674749) (Futahashi et al. 2012) reference sequences.

The Salamis anteva circular 15,201 bp mitogenome assembly was composed of 4040 paired reads with nucleotide composition: 40.5% A, 11.8% C, 7.6% G, and 39.9% T. The gene composition and order in the P. andremiaja mitogenome matches those of other butterflies (McCullagh and Marcus 2015). Precis andremiaja mitochondrial protein-coding genes use the start condons: ATG (COX2, ATP6, COX3, NAD4, NAD4L, CYTB, NAD1), ATT (NAD2, ATP8, NAD6), ATC (NAD3), ATA (NAD5), and CGA (COX1). The mitogenome contains three protein-coding genes (COX1, COX2, NAD4) with single-nucleotide (T) stop codons and one gene (NAD2) with a two-nucleotide (TA) stop codon completed by adding 3′ A residues post-transcriptionally. The NAD5 gene terminates with an unusual TAG stop codon. The mitochondrial transcription termination site in S. anteva is GTACTAAAT, while in most species in the tribe Junoniini the first and last positions of this binding site are adenine (A) bases. The locations and structures of tRNAs were determined using ARWEN v.1.2 (Laslett and Canback 2008). The mitochondrial rRNAs are typical of Lepidoptera (McCullagh and Marcus 2015) with characteristic cloverleaf secondary structures except for trnS (AGN) where a loop replaces the dihydrouridine arm.

Phylogenetic reconstructions used complete mitogenomes from S. anteva, 25 representatives from tribe Junonini, and 4 outgroup species from other tribes within the Nymphalinae (McCullagh and Marcus 2015; Peters and Marcus 2016; Peters and Marcus 2017; Lalonde and Marcus 2018; McCullagh and Marcus 2018). Sequences were aligned in CLUSTAL Omega (Sievers et al. 2011) and analyzed by parsimony and maximum likelihood (model selected by jModeltest 2.1.7 (Darriba et al. 2012) and likelihood ratio test (Huelsenbeck and Rannala 1997)) algorithms in PAUP* 4.0b8/4.0d78 (Swofford 2002) (Figure 1). Phylogenetic analysis places S. anteva as the sister-taxon to Yoma sabina within tribe Junoniini which is congruent with previous molecular phylogenetic analyses (Wahlberg et al. 2005; Kodandaramaiah and Wahlberg 2007).

Figure 1. Maximum likelihood phylogeny (GTR + G model, Gamma =0.1930, likelihood score 75924.15364) of Salamis anteva, 25 additional mitogenomes from tribe Junonini, and 4 outgroup species from other tribes in subfamily Nymphalinae based on 1 million random addition heuristic search replicates (with tree bisection and reconnection). One million maximum parsimony heuristic search replicates produced 24 equally most parsimonious trees (parsimony score 11461 steps) one of which is identical to the topology of the maximum likelihood tree. Numbers above each node are maximum likelihood bootstrap values and numbers below each node are maximum parsimony bootstrap values (each from 1 million random fast addition search replicates).

Acknowledgements

We thank Aleksandar Ilik and Debbie Tsuyuki (Children’s Hospital Research Institute of Manitoba Next Generation Sequencing Platform) for assistance with library preparation and sequencing.

Disclosure statement

The authors report no conflicts of interest and are solely responsible for this paper.

Additional information

Funding

This work received support from Natural Sciences and Engineering Research Council of Canada (NSERC) under Grants RGPIN386337-2011 and RGPIN-2016-06012.