The complete mitochondrial genome of the smudged eighty-eight butterfly Diaethria gabaza eupepla (Salvin & Godman, 1868) (Insecta: Lepidoptera: Nymphalidae)

Abstract The smudged eighty-eight butterfly Diaethria gabaza eupepla (Salvin & Godman, 1868) (Nymphalidae) is a vividly colored aposematic butterfly from Central and South America. A complete circular mitochondrial genome (mitogenome) of 15,156 bp from D. gabaza eupepla was assembled from a genome skimming Illumina sequence library. The AT-rich (80.5% AT) mitogenome consists of 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and a control region in the typical butterfly gene order. Diaethria gabaza eupepla COX1 begins with an atypical CGA start codon and ATP6, COX1, COX2, CYTB, ND1, ND4, ND4L, and ND5 mRNAs contain incomplete stop codons completed by the addition of 3’ A residues. Phylogenetic reconstruction places Diaethria as the sister clade to Hamadryas within monophyletic nymphalid subfamily Biblidinae, consistent with previous phylogenetic hypotheses.

The Living Prairie Mitogenomics Consortium is a structured inquiry exercise for undergraduates (Marcus et al. 2010) who assemble arthropod mitogenomes for improved DNA-based species identification and phylogenetics (Living Prairie Mitogenomics Consortium 2017, 2018, 2021. Participating students assembled, annotated, and analyzed sequence data (further curated by the instructor) and conducted a literature review for presentation here.
The butterflies of the genus Diaethria Billberg, 1820 (Nymphalidae: Biblidinae: Callicorini) are notable in that they possess color patterns on the ventral hind wings that resemble the numerals "88" or "89" and so are known by the common name "eighty-eight butterflies" (Dias et al. 2012). These distinctive patterns create a black, white, and red aposematic wing display intended to deter predators from eating these butterflies (Chai 1986;Pinheiro 1996). The genus includes a dozen species that occur between Texas, USA and Tierra del Fuego, Argentina (Dias et al. 2012). Male Diaethria are strongly attracted to urine-soaked sand and engage in mudpuddling behavior (DeVries 1987 A leg was removed from the specimen and DNA was prepared using a DNEasy Blood and Tissue kit (Qiagen, D€ usseldorf, Germany) with slight protocol modifications as described in McCullagh and Marcus (2015). DNA was sheared by sonication and a fragment library was prepared as previously described (Peters and Marcus 2017) using a NEBNext Ultra II DNA Library Prep Kit before sequencing by Illumina NovaSeq6000 (San Diego, CA) (Marcus 2018). Mitogenome assembly and annotation of the D. gabaza eupepla (GenBank accession MZ981736) was performed by mapping the resulting sequence library of 57,341,531 paired 150 bp reads (GenBank SRA PRJNA759138) to a Baeotus beotus reference mitogenome (Lepidoptera: Nymphalidae: Nympahlina: Coeini, MW566598 (Lalonde 2021)) using 5 iterations of the medium sensitivity settings of Geneious Prime 2021.1.1. Nuclear rRNA repeat sequences are increasingly recognized as being very useful for phylogenetic comparisons (Dodsworth 2015;Coissac et al. 2016;Marcus 2018;Krehenwinkel et al. 2019), so we also assembled the complete D. gabaza eupepla nuclear rRNA repeat (GenBank MZ981737) using a B. beotus (MW571038) (Lalonde 2021) reference sequence.
Phylogenetic reconstruction (Figure 1) was conducted using the complete mitogenome of D. gabaza eupepla and 31 other complete mitogenomes from the family Nymphalidae, including outgroup species Limenitis sydyi from nymphalid subfamily Limenitidinae and available from GenBank (Alexiuk et al. 2020;Hamilton et al. 2020;Lalonde and Marcus 2020;Payment et al. 2020;Alexiuk et al. 2021aAlexiuk et al. , 2021bLalonde 2021). GenBank accession numbers are listed in Figure 1. Mitogenome sequences were aligned in CLUSTAL Omega (Sievers et al. 2011) and analyzed using Bayesian Inference with the GTR þ I þ G model (model selected using jModeltest 2.1.1 (Darriba et al. 2012)) in Mr. Bayes version 3.2.7 (Ronquist and Huelsenbeck 2003;Ronquist et al. 2012). As expected based on a previous phylogenetic hypothesis (Wahlberg et al. 2009), phylogenetic analysis placed D. gabaza eupepla as the sister taxon to Hamadryas epinome in a monophyletic clade with mitogenomes from nymphalid subfamily Biblidinae.

Acknowledgments
Thanks to Melanie Lalonde and Josephine Payment for assistance with DNA extraction and for bioinformatics pipeline development. The authors thank Genome Quebec for assistance with library preparation and sequencing.

Disclosure statement
No potential conflict of interest was reported by the authors.

Author contributions
Jeffrey Marcus was responsible for the conception and design of the study as well as the initial drafting of the paper. All of the authors were involved in the analysis and interpretation of the data, revising the manuscript critically for intellectual content, and gave the final approval of the version to be published. All authors agree to be accountable for all aspects of the work.

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at [https://www.ncbi.nlm.nih.gov] (https://www.ncbi.nlm.nih.gov/) under the accession no. MZ981736-MZ981737. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA759138, SRX11982685, and SAMN21156917 respectively.