Characterization of the complete mitochondrial genome of an endemic species in China, Aulocera merlina (Lepidoptera: Nymphalidae: Satyrinae) and phylogenetic analysis within Satyrinae

Abstract The mitochondrial genome (mitogenome) has been extensively used as molecular markers in determining the insect phylogenetic relationships. In order to resolve the relationships among tribes and subtribes of Satyrinae at the mitochondrial genomic level, we obtained the complete mitogenome of Aulocera merlina (Oberthür, 1890) (Lepidoptera: Nymphalidae: Satyrinae) with a size of 15,259 bp. The mitogenome consisted of 37 typical genes, including 13 protein‐coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an A + T‐rich region. The gene organization and arrangement were similar to those of all other known Satyrinae mitogenomes. All PCGs were initiated with the canonical codon pattern ATN, except for the cox1 gene, which used an atypical CGA codon. Nine PCGs used the complete stop codon TAA, while the remaining PCGs (cox1, cox2, nad4, and nad5) were terminated with a single T nucleotide. The canonical cloverleaf secondary structures were found in all tRNAs, except for trnS1 which lacked a dihydrouridine arm. The 448 bp A + T‐rich region was located between rrnS and trnM, and it included the motif ATAGA followed by a 19‐bp poly‐T stretch and a microsatellite‐like (TA)6 element preceded by the ATTTA motif. The phylogenetic tree, inferred using Bayesian inference and maximum likelihood methods, generated similar tree topologies, revealing well‐supported monophyletic groups at the tribe level and recovering the relationship ((Satyrini + Melanitini) + ((Amathusiini + Elymniini) + Zetherini)). The close relationship between Satyrina and Melanargiina within the Satyrini was widely accepted. Additionally, Lethina, Parargina, and Mycalesina were closely related and collectively formed a sister group to Coenonymphina. Moreover, A. merlina was closely related to Oeneis buddha within the Satyrina. These findings will provide valuable information for future studies aiming to elucidate the phylogenetic relationships of Satyrinae.

Therefore, it is crucial to accurately identify Satyrinae species and understand their evolutionary relationships for related studies.
Given the complexity of these relationships, it is essential to utilize mitochondrial genome (mitogenome) sequences of these species for taxonomic and phylogenetic analyses.
Recent studies have also shown that the entire mitogenome can provide abundant information for resolving phylogenetic relationships within Lepidoptera at various hierarchical levels (Boore, 2006;Li et al., 2021;Qin et al., 2019;Wu et al., 2014Wu et al., , 2022)).Therefore, it is important to study more taxa and mitogenomes to gain a better understanding of the internal relationships of Satyrinae.
Aulocera merlina (Oberthür, 1890), an endemic species in China, belongs to the subfamily Satyrinae and is mainly distributed in Sichuan, Yunnan, and other areas (Chou, 2000).Although this species is similar in appearance to A. padma, it is smaller in size.However, Aulocera has not been included in previous studies on the phylogeny of Satyrinae using mitogenome sequences.In this study, we determined the complete mitogenome of A. merlina and compared it with other known mitogenomes of Satyrinae species for the first time.
Additionally, we reconstructed phylogenetic trees based on the available mitogenome sequences, including the newly sequenced mitogenome, to gain insight into the phylogenetic relationships among the major lineages of the Satyrinae.

| Sampling and DNA extraction
The adult individuals of A.merlina were collected from Lijiang City, Yunnan Province, P. R. China, in July 2016.They were identified based on morphological characteristics.After collection, the samples were immediately preserved in 95% ethanol and stored at −80°C under the Voucher number SMU-20160726.Total genomic DNA was extracted from thorax muscle tissues of a single specimen using the Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China).The extracted DNA was then used for 500-bp library construction using the NEBNext Ultra DNA Library Prep Kit for Illumina sequencing.

| Mitogenome sequencing and assembly
Sequencing was carried out on the Illumina NovaSeq 6000 platform (BIOZERON Co., Ltd., Shanghai, China).Approximately, 13841.3Mb of raw data from A.merlina were generated with 150 bp pairedend read lengths.De novo assembly with GetOrganelle v1.6.4 (Jin et al., 2020) referencing mitogenome of closely related species Minois dryas (GenBank accession number: NC_046591) (Shi et al., 2019) produced contigs of mitogenome.A number of potential mitochondrion reads were extracted from the pool of Illumina reads using BLAST searches against mitogenomes of related species M. dryas and the GetOrganelle result.The mitochondrion Illumina reads were obtained to perform mitogenome de novo assembly using the SPAdes−3.13.0 package (Nurk et al., 2017).The GetOrganelle assembly contig was optimized by the scaffolds from SPAdes-3.13.0 result.Finally, the assembled sequence was reordered and oriented according to the reference mitogenome, thus generating the final assembled mitochondrion genomic sequence.

| Mitogenome annotation and analyses
The mitogenome of A. merlina was annotated using the online MITOS tool (http:// mitos.bioinf.uni-leipz ig.de/ index.py) with default Aulocera merlina, Lepidoptera, mitochondrial genome, phylogeny, Satyrinae  (Continues) parameters (Bernt et al., 2013).Protein-coding genes (PCGs) and rRNA genes were annotated by aligning them with the homologous sequence from M. dryas, based on the invertebrate mitochondrial genetic code.The Tandem Repeats Finder program (http:// tandem.bu.edu/ trf/ trf.html) was utilized to predict the tandem repeats of the control region using the default parameters (Benson, 1999).

| Phylogenetic analyses
Phylogenetic analyses were conducted based on the A. merlina and 63 other available and complete mitogenome sequences of Satyrinae species from GenBank.Two species, Polyura nepenthes (Charaxinae) and Calinaga davidis (Calinaginae), were selected as outgroups (Table 1).The 13 PCGs and two rRNAs were first aligned individually using MEGA 11.0 software (Tamura et al., 2021) and then concatenated using DAMBE7 (Xia, 2018) for phylogenetic analyses.The best model (GTR + I + G) for concatenated sequences, determined by the corrected Akaike Information Criterion using jModeltest 2.1.10( Darriba et al., 2012), was selected.Maximum likelihood (ML) phylogenetic analysis was performed using IQ-TREE software (Nguyen et al., 2015).Bootstrap support (BS) values were evaluated using 1000 ultrafast bootstrap replicates (Hoang et al., 2018).Bayesian inference (BI) analysis was performed using MrBayes 3.2 (Ronquist et al., 2012).Four simultaneous Markov chains were run for 20 million generations, and trees were sampled every 100 generations.A Note: The specie with newly sequenced mitogenome was emphasized in bold. a The mitochondrial genome of the indicated species is incomplete.
| 5 of 16 burn-in of 25% was applied, and the remaining samples were used to generate a consensus tree and estimate the posterior probabilities (PP).The topologies of the phylogenetic trees were visualized using FigTree v1.4.2 (Rambaut, 2014).

| Mitogenome organization
The complete mitogenome of A. merlina was a typical circular DNA molecule of 15,259 bp in size (GenBank accession number: NC_068667).It consisted of 37 genes, including 13 PCGs, 22 tRNAs, and 2 rRNAs, and an A + T-rich region (Figure 1, Table 2).The gene order and arrangement of the newly determined mitogenome were similar to those of other butterflies.Among the genes, 23 genes (9 PCGs and 14 tRNAs) were located on the majority strand (J-strand), while the remaining 14  3).The A. merlina mitogenome contained a total of 12 intergenic spacers, ranging in size from 1 to 53 bp, with a total length of 84 bp.The longest intergenic spacer region was located between the genes of nad2 and trnW.Additionally, there were 10 overlapping regions (29 bp in total) in the mitochondrion, ranging in length from 1 to 8 bp.The longest overlapping region was found between the trnC and trnY genes (Table 2).

| Protein-coding genes and codon usage
The length of the 13 PCGs in the A. merlina mitogenome ranged from 165 bp for atp8 to 1735 bp for nad5, totaling 11,203 bp.Among these F I G U R E 1 Circular maps of the Aulocera merlina mitogenome.Gene names on the outside of the circle indicated that these genes are located on the majority strand, whereas the others are located on the minority strand.
Additionally, we calculated the values of Ka, Ks, and Ka/Ks for the 13 PCGs from 64 Satyrinae species.The results (Figure 3b) indicated that the Ka/Ks ratios were low and ranged from 0.043 (cox1) to 0.266 (atp8), suggesting that these genes underwent purifying selection (Meiklejohn et al., 2007).Consequently, the 13 PCGs are suitable for investigating phylogenetic relationships within the Satyrinae.

| A + T-rich region
The A + T-rich region of the A. merlina mitogenome spanned 448 bp and was situated between rrnS and trnM (Figure 1, Table 2).This region had the highest A + T content (92.0%) and a negative AT-skew (−0.020) (Table 3), while the GC-skew value was zero, indicating an equal proportion of G and C bases.The A + T-rich region also contained conserved structures commonly found in lepidopteran mitogenomes, such as the 'ATAGA' motif followed by a 19 bp poly-T stretch and a microsatellite-like (TA) 6 element preceded by the 'ATTTA' motif (Figure 5).Overall, negative AT skews and GC skews are common in Satyrinae.

| Phylogenetic relationships
Furthermore, we observed a 7 bp overlap (ATGATAA) between atp8 and atp6, which has been commonly found in other Lepidoptera species (Liu et al., 2018;Lu et al., 2018;Zhu et al., 2013).All PCGs were initiated by the typical ATN codon, except for cox1, which started with the unusual CGA codon, as seen in most other Satyrinae mitogenomes (Shi et al., 2019;Yang et al., 2020).Nine PCGs used TAA as the termination codon, while four PCGs (cox1, cox2, nad5, and nad4) terminated with an incomplete T codon.Incomplete termination codons of PCGs are commonly observed in Lepidoptera mitogenomes (Chen et al., 2020;Liu et al., 2018Liu et al., , 2023) ) and are converted into TAA by post-transcriptional polyadenylation (Ojala et al., 1981).The canonical cloverleaf secondary structures were found in all tRNAs, except in trnS1, which lacked the DHU arm, as observed in other reported nymphalids (Wu et al., 2014;Yang et al., 2020).found in lepidopterans, such as the ATAGA motif followed by a 19 bp poly-T stretch, and a microsatellite-like (TA) 6 element preceded by the ATTTA motif (Kim et al., 2014;Salvato et al., 2008).
To better understand their evolutionary relationships, we conducted a preliminary investigation using mitogenomic data.The In our ML and BI analyses, the robust phylogenetic relationships among the four tribes were described as ((Satyrini + Melanitini) + ((Amathusiini + Elymniini) + Zetherini)).The close relationship between Amathusiini and Elymniini was consistent with previous mitogenomic studies (Sun et al., 2021;Wu et al., 2022;Yang et al., 2020) and the BI analysis of Peña and Wahlberg (2008).
However, some studies regarded Elymniini as closer to Melanitini (Wahlberg et al., 2009;Yang & Zhang, 2015).Therefore, the tribe-level relationships remain undefined and require further validation with a more extensive sampling of taxa.
However, numerous challenges persist in resolving the systematics of Satyrini.The monophyly of Satyrini is strongly supported in the present study.Despite the limited taxon sampling within the diverse Satyrini, the Satyrina consistently appeared as a sister group to Melanargiina with strong support, which aligns with previous studies (Dan et al., 2021;Sun et al., 2021;Yang et al., 2020).Additionally, close relationships among three subtribes (Parargina, Lethina, and Mycalesina) received robust support, especially in our BI analysis, and the same pattern also recovered in previous mitogenomic studies (Dan et al., 2021;Sun et al., 2021;Wu et al., 2022;Yang et al., 2020) and multiple-locus investigations (Peña & Wahlberg, 2008;Wahlberg et al., 2009;Yang & Zhang, 2015).Our phylogenetic results also confirmed the closer relationship between O. urda and Davidina, as previously reported (Dan et al., 2021;Lukhtanov & Dubatolov, 2020;Usami et al., 2021).Unfortunately, Lethina was not found to be a F I G U R E 8 Scatter plot of AT and GC skews in the Satyrinae species.

F
Gene variation of 13 PCGs in Satyrinae.(a) The sliding window analysis shows the value of nucleotide diversity.(b) The Ka, Ks, and Ka/Ks of each PCG among Satyrinae representatives.Ka-nonsynonymous substitution; Ks-synonymous substitution.
).The monophyly of Satyrini was well supported in both ML and BI analyses (BS = 100%, PP = 1.00).Moreover, Satyrini clustered with Melanitini as a sister group with strong support in the BI tree (PP = 0.96), but medium support in the ML tree (BS = 73%).Additionally, Amathusiini was identified as the sister group to Elymniini with a strong node support value (BS = 99%, PP = 1.00).The phylogenetic relationships among the five tribes of Satyrinae were found to be the same and arranged as follows: ((Satyrini + Melanitini) + ((Amathusiini + Elymniini) + Zetherini)).Within the Satyrini, except for Lethina, the subtribes Ypthima, Mycalesina, Parargina, Coenonymphina, Satyrina, Melanargiina, Maniolina, and Erebiina were monophyletic with strong support in our analyses.The relationships among the nine subtribes of Satyrini obtained from the ML and BI analyses were highly similar, although some differences exist.The BI tree showed that two well-supported phylogenetic clades were identified from the nine sampled subtribes: clade I including Parargina, Mycalesina, Lethina, and Coenonymphina (PP = 0.99), and the remaining five subtribes constituting clade II (PP = 1.00).Furthermore, Ypthimina formed a monophyletic group with strong support (PP = 1.00) within clade II, and the phylogenetic relationships among the five subtribes recovered herein were ((Ypthimina + (Maniolina + Erebiina)) + (Satyrina + Melanargiina)) with strong support in the BI tree.The ML analysis indicated that the nine sampled subtribes of the Satyrini were also split into two clades as detected in our BI tree.However, most of the subtribe-level relationships had weak support in the ML tree.The monophyly of the genus Lethe was well supported and was separated from Lethina in both ML and BI trees (BS = 98%, PP = 1.00).Furthermore, the ML analysis suggested that Ninguta schrenckii was clustered with three species of the genus Neope within Lethina, although with weak support (BS = 55%).However, in the BI analysis, N. schrenckii was positioned as the sister group to the grouping of the Parargina, Mycalesina, and the genus Lethe, but with weak support (PP = 0.36).Interestingly, both our ML and BI analyses indicated a close relationship between A. merlina and Oeneis buddha within the subtribe Satyrina, which was sister to Melanargiina with high values (BS = 100%, PP = 1.00).F I G U R E 5The features of A + T-rich region in the Aulocera merlina mitogenome.autonoe(Dan et al., 2021) and the GC-skew ranging from −0.271 in Stichophthalma louisa to −0.153 in Callerebia polyphemus (Figure8).
The A + Trich region displayed several structural characteristics commonly F I G U R E 7 Phylogenetic tree inferred by Bayesian inference method based on a concatenated matrix of 13 PCGs and 2 rRNAs.Bayesian posterior probabilities (PP) are shown at relevant branches.

TA B L E 1
List of taxa analyzed in this study together with relevant information.
Characteristics of the Aulocera merlina mitogenome.Strand of the genes is presented as J for majority and N for minority strand.In the column for intergenic length, a positive sign indicates the interval in base pairs between genes, while the negative sign indicates overlapping base pairs between genes.Nucleotide composition and skewness of the Aulocera merlina mitogenome.
TA B L E 2 TA B L E 3