Characterization of the complete mitochondrial genome of the Hypolimnas misippus Linnaeus 1764 (Lepidoptera: nymphalidae)

Abstract In this study, we sequenced the complete mitochondrial genome (mitogenome) of Hypolimnas misippus Linnaeus 1764, which is 15,283 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and an adenine (A) + thymine (T)-rich (D-loop) region. The overall GC level is 19.8%. The phylogenetic position of H. misippus was evaluated using 48 previously published complete mitogenomes, and the results reveals that H. misippus is most closely related to H.bolina.


Introduction
The subfamily Nymphalinae (Lepidoptera: Nymphalidae) comprises about 500 species distributed around the world.Several species in the group have been used as model organisms in ecological and evolutionary studies (Brower et al. 2000;Boggs et al. 2003;Kemp et al. 2005).Despite the relatively rich amounts of basic studies about this butterfly group, there is no complete mitochondrial genomes publicly available for Hypolimnas misippus Linnaeus.1764.Therefore, we sequenced the complete mitochondrial DNA genome of H. misippus to provide baseline data for this species and also to better understand its relationship within the subfamily Nymphalinae.

Sample collection and preservation
Specimens of female H. misippus (Figure 1) were collected with hand nets from South China Botanical Garden (23 18 0 35 00 N, 113 36'56 00 E), Guangzhou, China.The wings of female butterflies are orange-yellow, and with a small white spot at the top corner.Two rows of small white spots in pairs are arranged on the outer edge of both wings.The color of the back side of the wings is pale, and there are 3 white spots above the middle chamber.There is one black spot on the middle of the anterior margin of the hindwing and one outside the end of the middle wing vein (Chou 1998).

DNA extraction, sequencing, and assembly
Total genomic DNA was extracted from the thorax muscle of a single individual butterfly using the Sangon Animal genome DNA Extraction Kit (Shanghai, China).The voucher specimen was deposited at Southwest Forestry University under the voucher number SCBGJBJD0916 (Jin Xu, xujin2798@163.com).After DNA isolation, 1 lg of purified DNA was fragmented and used to construct short-insert libraries (insert size $350 bp) according to the manufacturer's instructions (BIG-500).Then DNA libraries were sequenced by Guangzhou BIO&DATA Biotechnologies Inc. (Guangzhou, China) on the BGI-500 Sequencing System (BGI, Shenzhen, China) using PE 150 bp reads.The filtered sequences were assembled using the SPAdes assembler 3.10.0(Bankevich et al.2012).The coverage depth (600 Â $ 4060 Â, mean: 2709.9Â, Figure S1)  was calculated by Geneious R11 (Kearse et al. 2012) by mapping the total clean reads to de novo assembled plastome.

Annotation and analysis
The mito-genome was annotated using MITOS2 (Donath et al.2019), BLASTþ (Camacho et al. 2009) and tRNAscan (Schattner et al. 2005).To investigate its taxonomic relationships, a maximum likelihood (ML) phylogeny was reconstructed based on the whole mitogenome of 46 Nymphalinae butterflies and two outgroup butterflies (Neptis alwina and Parthenos sylvia).All sequences were aligned using the program MAFFT version 7.471 (Katoh and Standley 2013), and then a maximum-likelihood (ML) phylogenetic tree was constructed using FastTree version 2.1.10with Generalized Time-Reversible (GTR) model, statistical support for branches was tested by Shimodaira-Hasegawa (SH) test (Price 2010).

Phylogenetic analysis
The phylogenetic position of H. misippus was inferred by a ML phylogenetic tree using FastTree based on the whole mitogenome of 46 Nymphalinae butterflies and two outgroup butterflies (Neptis alwina and Parthenos sylvia) (Wu et al. 2014;Chen et al. 2020;Hamilton et al. 2020;Liu et al. 2020;Li et al. 2020;Alexiuk et al. 2021;Lalonde 2021;Lohse et al. 2021;Aguila et al. 2021).As shown in Figure 2, the ML phylogenetic tree shows that H. misippus was most closely related to H. bolina, with support values of 100%.Furthermore, the genus Hypolimnas were clustered together with Precis andremiaja.This relationships is congruent with previous phylogenetic studies (Kim et al. 2021).

Conclusion
The complete mitochondrial genome of H. misippus was sequenced on the BIG-500 platform to generate a 15,283 bp mitogenome (Genbank accession no.OL711673).The phylogenetic position of H. misippus within the subfamily of Nymphalinae was determined, and the results showed that H. misippus was closely related to H. bolina.Our findings are helpful to understand the phylogenetic status of H. misippus.
It also provides baseline molecular data for future studies on the evolutionary relationships within the Nymphalidae.

Ethical approval
This study does not involve Endangered or protected species according to IUZN (2021).The approval of sample collection is not required according to the Animal Ethical and Welfare Committee of Southwest Forestry University.

Figure 3 .
Figure 3. Maximum-likelihood phylogenetic tree based on whole mitogenome from 46 Nymphalinae butterfly and two outgroup butterfly (Neptis alwina and Parthenos sylvia) and the support values are shown at the branches.