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. 2024 Apr 30;14(5):e11355. doi: 10.1002/ece3.11355

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

Qinghui Shi 1,✉, Jinling Xie 1, Jialing Wu 1, Shengchung Chen 1, Gang Sun 1, Juncheng Zhang 1,2
PMCID: PMC11061544  PMID: 38694754

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.

Keywords: Aulocera merlina, Lepidoptera, mitochondrial genome, phylogeny, Satyrinae

In this paper, the complete mitochondrial genome sequence of an endemic species in China, Aulocera merlina (Lepidoptera: Nymphalidae: Satyrinae) was determined and compared with that of other known mitogenomes of Satyrinae species. Moreover, the phylogenetic trees were reconstructed based on the available mitogenome sequences, including the newly sequenced mitogenome, to gain a better understanding of the phylogenetic relationships among the major lineages of the Satyrinae.

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1. INTRODUCTION

The Satyrinae, a subgroup within the Nymphalidae family, is known for its high diversity, comprising over 2500 species found across all continents except Antarctica (Ackery et al., 1999; Peña & Wahlberg, 2008). This diversity has made Satyrinae species popular model organisms in various research fields including ecology (Schmitt & Haubrich, 2008), developmental biology (Oliver et al., 2012), and conservation biology (Slamova et al., 2013). Therefore, it is crucial to accurately identify Satyrinae species and understand their evolutionary relationships for related studies. Currently, there are nine tribes and 16 subtribes defined within the Satyrinae (Marín et al., 2011). However, the phylogenetic relationships at both the tribe and subtribe levels are still not fully resolved (e.g., Chen et al., 2020; Dan et al., 2021; Marín et al., 2011; Peña et al., 2006; Peña & Wahlberg, 2008; Sun et al., 2021; Wahlberg et al., 2009; Wu et al., 2022; Yang et al., 2020; Yang & Zhang, 2015). Given the complexity of these relationships, it is essential to utilize mitochondrial genome (mitogenome) sequences of these species for taxonomic and phylogenetic analyses.

The insect mitogenome is a circular, double‐stranded molecule that encodes a highly conserved set of 37 genes. These genes include 13 protein‐coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes (rRNAs, rrnL, and rrnS) (Boore, 1999; Cameron, 2014). Additionally, it typically contains an A + T‐rich region that includes essential regulatory elements for replication and transcription (Taanman, 1999). Mitogenomic sequences have been widely used as molecular markers in phylogenetic analysis, comparative genomics studies, and species identification due to unique features such as strict maternal inheritance, rapid mutation rate, and limited recombination (e.g., Avise, 2009; Cameron, 2014; Liu et al., 2023; Qin et al., 2019; Tyagi et al., 2020; Yan et al., 2023). 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., 2014, 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.

2. MATERIALS AND METHODS

2.1. 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.

2.2. Mitogenome sequencing and assembly

Sequencing was carried out on the Illumina NovaSeq 6000 platform (BIOZERON Co., Ltd., Shanghai, China). Approximately, 13841.3 Mb of raw data from A.merlina were generated with 150 bp paired‐end 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.

2.3. Mitogenome annotation and analyses

The mitogenome of A. merlina was annotated using the online MITOS tool (http://mitos.bioinf.uni‐leipzig.de/index.py) with default 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). The circular mitogenomic map was generated using the CGview server (http://stothard.afns.ualberta.ca/cgview_server/) (Grant & Stothard, 2008). Secondary structures for tRNAs were manually illustrated using Adobe Illustrator 2021 based on the MITOS predictions. The nucleotide composition and relative synonymous codon usage (RSCU) values were analyzed using MEGA 11.0 software (Tamura et al., 2021). The AT and GC asymmetry were represented by the values of AT‐skew and GC‐skew, calculated using the following formulas: AT‐skew = (A − T)/(A + T) and GC‐skew = (G − C)/(G + C) (Perna & Kocher, 1995). Nucleotide diversity and the ratio of nonsynonymous substitution (Ka) to synonymous substitution (Ks) for PCGs were calculated using DNASP 6.0 (Julio et al., 2017).

2.4. 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 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).

TABLE 1.

List of taxa analyzed in this study together with relevant information.

Subfamily Tribe/subtribe Species Size (bp) Accession number Reference
Satyrinae (ingroup) Satyrini/Lethina Lethe chandica 15,206 MZ501804 Yan et al. (2023)
Lethe verma 15,239 NC_050916 Chen et al. (2020)
Lethe dura 15,259 KF906485 Unpublished
Lethe uemurai 15,272 NC_050915 Chen et al. (2020)
Lethe sicelis 15,239 LC541741 Nagata et al. (2020)
Lethe titania 15,257 NC_050914 Chen et al. (2020)
Lethe syrcis 15,252 NC_050913 Chen et al. (2020)
Lethe satyrina 15,271 NC_050912 Chen et al. (2020)
Lethe oculatissima 15,243 NC_050911 Chen et al. (2020)
Lethe nigrifascia 15,239 NC_050910 Chen et al. (2020)
Lethe marginalis 15,229 NC_050909 Chen et al. (2020)
Lethe helle 15,253 NC_050908 Chen et al. (2020)
Lethe hayashii 15,246 NC_050907 Chen et al. (2020)
Lethe baucis 15,251 NC_050906 Chen et al. (2020)
Lethe baileyi 15,225 NC_050905 Chen et al. (2020)
Lethe albolineata 15,248 NC_028507 Li et al. (2016)
Lethe confusa a 14,945 MT654529 Unpublished
Ninguta schrenckii 15,261 KF881052 Fan et al. (2016)
Neope goschkevitschii 15,286 LC541740 Nagata et al. (2020)
Neope pulaha 15,209 KF590543 Wu et al. (2014)
Neope muirheadii 15,217 MN242789 Yang et al. (2020)
Satyrini/Ypthimina Ypthima akragas 15,227 NC_024420 Wu et al. (2014)
Ypthima motschulskyi 15,232 MN242788 Yang et al. (2020)
Ypthima baldus 15,304 NC_056106 Li et al. (2020)
Argestina inconstans 15,219 NC_079673 Unpublished
Argestina pomena 15,226 NC_070116 Unpublished
Callerebia polyphemus 15,156 NC_058609 Yan et al. (2023)
Callerebia suroia 15,208 KF906483 Shi et al. (2016)
Satyrini/Mycalesina Mycalesis intermedia 15,386 MN610565 Wu et al. (2020)
Mycalesis mineus 15,267 NC_025762 Tang et al. (2014)
Mycalesis francisca 15,279 MN242790 Yang et al. (2020)
Bicyclus anynana 16,129 OX359232 Unpublished
Satyrini/Parargina Lopinga achine 15,284 MT117843 Wu et al. (2022)
Pararge aegeria 15,240 KJ547676 Teixeira da Costa (2016)
Lasiommata deidamia 15,244 MG880214 Sun et al. (2021)
Lasiommata majuscula 15,263 MN012997 Liu et al. (2020)
Lasiommata megera 15,282 OV743337 Liu et al. (2020)
Satyrini/Coenonymphina Coenonympha amaryllis 15,125 NC_046491 Zhou, Yang, et al. (2020)
Coenonympha tullia a 15,316 KM592972 Unpublished
Triphysa phryne 15,143 NC_024551 Zhang et al. (2016)
Satyrini/Satyrina Aulocera merlina 15,259 NC_068667 This study
Davidina armandi 15,214 NC_028505 Unpublished
Hipparchia autonoe 15,435 OK094488 Dan et al. (2021)
Hipparchia semele 15,223 OW121739 Unpublished
Minois dryas 15,195 NC_046591 Shi et al. (2019)
Oeneis urda 15,248 NC_046889 Zhou, Liang, et al. (2020)
Oeneis buddha 15,259 OK094489 Dan et al. (2021)
Paroeneis palaearcticus 15,942 OK094490 Dan et al. (2021)
Satyrini/Melanargiina Melanargia asiatica 15,142 NC_024550 Huang et al. (2016)
Melanargia caoi 15,469 MN012999 Liu et al. (2020)
Melanargia meridionalis 15,442 NC_067761 Unpublished
Melanargia galathea 15,367 OV049880 Vila et al. (2022)
Satyrini/Maniolina Aphantopus hyperantus 15,240 KM592969 Unpublished
Maniola jurtina 15,258 HG995237 Lohse and Weir (2021)
Satyrini/Erebiina Erebia aethiops 15,201 OV281099 Lohse and Lohse (2022)
Erebia ligea 15,196 OU785248 Lohse et al. (2022)
Melanitini Melanitis phedima 15,142 KF590538 Wu et al. (2014)
Melanitis leda 15,122 JF905446 Shi et al. (2013)
Amathusiini Stichophthalma louisa 15,721 KP247523 Unpublished
Stichophthalma howqua a 14,020 KF990129 Shi et al. (2015)
Stichophthalma camadeva 15,257 OQ942883 Unpublished
Elymniini Elymnias hypermnestra 15,167 KF906484 Shi et al. (2015)
Elymnias malelas 15,161 OQ942881 Unpublished
Zetherini Ethope himachala 15,531 NC_070168 Unpublished
Charaxinae (outgroup) Charaxini Polyura nepenthes 15,333 NC_026073 Shi et al. (2015)
Calinaginae (outgroup) – Calinaga davidis 15,267 NC_015480 Xia et al. (2011)
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Note: The specie with newly sequenced mitogenome was emphasized in bold.

a

The mitochondrial genome of the indicated species is incomplete.

3. RESULTS

3.1. 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 genes (4 PCGs, 8 tRNAs, and 2 rRNAs) were transcribed on the minority strand (N‐strand). The nucleotide composition of A. merlina was significantly biased toward A + T, with a value of 79.9%. The nucleotide skewness statistics for the mitogenome showed slight T skews (−0.031) and moderate C skews (−0.224) (Table 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).

FIGURE 1.

FIGURE 1

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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.

TABLE 2.

Characteristics of the Aulocera merlina mitogenome.

Gene Coding strand Location Size (bp) Anticodon Intergenic nucleotides Start codon Stop codon
trnM J 1–69 69 CAU – – –
trnI J 70–133 64 GAU 0 – –
trnQ N 131–199 69 UUG −3 – –
nad2 J 253–1266 1014 – 53 ATT TAA
trnW J 1265–1331 67 UCA −2 – –
trnC N 1324–1387 64 GCA −8 – –
trnY N 1388–1452 65 GUA 0 – –
cox1 J 1458–2988 1531 – 5 CGA T
trnL2 (UUR) J 2989–3055 67 UAA 0 – –
cox2 J 3056–3731 676 – 0 ATG T
trnK J 3732–3802 71 CUU 0 – –
trnD J 3804–3869 66 GUC 1 – –
atp8 J 3870–4034 165 – 0 ATC TAA
atp6 J 4028–4705 678 – −7 ATG TAA
cox3 J 4705–5493 789 – −1 ATG TAA
trnG J 5496–5562 67 UCC 2 – –
nad3 J 5563–5916 354 – 0 ATT TAA
trnA J 5916–5979 64 UGC −1 – –
trnR J 5981–6042 62 UCG 1 – –
trnN J 6045–6111 67 GUU 2 – –
trnS1 (AGN) J 6109–6168 60 GCU −3 – –
trnE J 6170–6235 66 UUC 1 – –
trnF N 6236–6301 66 GAA 0 – –
nad5 N 6302–8036 1735 – 0 ATT T
trnH N 8037–8100 64 GUG 0 – –
nad4 N 8101–9439 1339 – 0 ATG T
nad4l N 9439–9726 288 – −1 ATA TAA
trnT J 9732–9795 64 GUG 5 – –
trnP N 9796–9860 65 UGG 0 – –
nad6 J 9863–10,387 525 – 2 ATT TAA
cob J 10,387–11,538 1152 – −1 ATG TAA
trnS2 (UCN) J 11,545–11,609 65 UGA 6 – –
nad1 N 11,608–12,564 957 – −2 ATG TAA
trnL1 (CUN) N 12,566–12,632 67 UAG 1 – –
rrnL N 12,638–13,972 1335 – 5 – –
trnV N 13,973–14,037 65 UAC 0 – –
rrnS N 14,038–14,811 774 – 0 – –
A + T‐rich region 14,812–15,259 448 – 0
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Note: 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.

TABLE 3.

Nucleotide composition and skewness of the Aulocera merlina mitogenome.

Feature Size (bp) Nucleotide frequency (%) AT‐Skew GC‐Skew
A T G C A + T G + C
Whole genome 15,259 38.7 41.2 7.8 12.3 79.9 20.1 −0.031 −0.224
Protein‐coding genes 11,203 32.7 45.7 11.0 10.7 78.4 21.7 −0.166 0.014
1st codon position 3724 36.2 36.8 16.2 10.8 73.0 27.0 −0.008 0.200
2nd codon position 3724 21.6 48.7 13.5 16.2 70.3 29.7 −0.385 −0.091
3rd codon position 3724 40.0 51.5 3.3 5.2 91.5 8.5 −0.126 −0.224
tRNA genes 1444 41.1 39.5 11.3 8.0 80.6 19.3 0.020 0.171
rRNA genes 2109 46.2 38.7 10.1 5.0 84.9 15.1 0.088 0.338
A + T‐rich region 448 45.1 46.9 4.0 4.0 92.0 8.0 −0.020 0
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3.2. 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 PCGs, nine genes (cox1, cox2, cox3, atp6, atp8, nad2, nad3, nad6, and cob) were encoded by the majority strand, while the remaining four genes (nad1, nad4, nad4l, and nad5) were encoded by the minority strand (Figure 1, Table 2). All PCGs were initiated with the conventional ATN codons, except for cox1 which started with the atypical CGA codon. Nine out of the 13 PCGs ended with the typical stop codon (TAA), while the remaining PCGs (cox1, cox2, nad4, and nad5) concluded with an incomplete termination codon (T‐‐). The 13 PCGs also exhibited a significant bias toward A + T content (78.4%). Notably, the third codon positions had a considerably higher A + T content (91.5%) compared to the first (73.0%) and second positions (70.3%) (Table 3). The mitogenome of A. merlina encodes 3724 amino acids (excluding stop codons). The relative synonymous codon usage (RSCU) values for the 13 PCGs indicated that the five most frequently used codons in the mitogenome of A. merlina were UUA (L), UCU (S), GCU (A), CCU (P), and GGA (G) (Figure 2a). Leucine (L, Leu) was the most commonly encoded amino acid (12.7%), followed by isoleucine (I, Ile, 12.5%), phenylalanine (F, Phe, 10.1%), methionine (M, Met, 6.9%), and asparagine (N, Asn, 6.8%). On the other hand, cysteine (C, Cys) was the least common amino acid (1.0%) (Figure 2b).

FIGURE 2.

FIGURE 2

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Codon usage of the Aulocera merlina mitogenome. Codon families are provided on the X‐axis. (a) Relative synonymous codon usage (RSCU). (b) Codon distribution.

To assess the variation patterns of the 13 PCGs of Satyrinae, nucleotide diversity was calculated through sliding window analysis for each PCG. The results revealed that cox2 was the most conserved (Pi = 0.103), while nad3 exhibited the highest variability (Pi = 0.162) (Figure 3a). The gene with the next highest nucleotide diversity after nad3 was nad6 (Pi = 0.159), followed by atp8 (Pi = 0.144), cox3 (Pi = 0.136), cob (Pi = 0.133), and nad4 (Pi = 0.132). 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.

FIGURE 3.

FIGURE 3

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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.

3.3. Transfer and ribosomal RNA genes

The A. merlina mitogenome was found to contain 22 tRNA genes, ranging in size from 60 bp (trnS(AGN)) to 71 bp (trnK) (Table 2). Out of the 22 tRNA genes, 14 were located on the majority strand (trnA, trnE, trnD, trnG, trnK, trnI, trnL2, trnM, trnN, trnR, trnS1, trnS2, trnT, and trnW), while the remaining eight were embedded in the minority strand (trnQ, trnC, trnY, trnF, trnH, trnP, trnL1, and trnV) (Figure 1, Table 2). The total length of the 22 tRNAs was 1444 bp, and the A + T content was 80.6% with positive AT‐skew (0.020) and GC‐skew (0.171). All tRNA genes exhibited a typical cloverleaf secondary structure, except for trnS1, which lacks the dihydrouridine (DHU) stem arm (Figure 4). The A. merlina mitogenome also contained two rRNA genes, both of which were embedded in the minority strand. The larger ribosomal RNA (rrnL) was found between trnL1 and trnV, while the smaller ribosomal RNA (rrnS) was located between trnV and the A + T‐rich region (Figure 1, Table 2). The lengths of rrnL and rrnS were 1335 bp (A + T content 84.7%) and 774 bp (A + T content 85.2%), respectively (Table 3).

FIGURE 4.

FIGURE 4

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Predicted secondary cloverleaf structures for the tRNAs of the Aulocera merlina.

3.4. 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).

FIGURE 5.

FIGURE 5

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The features of A + T‐rich region in the Aulocera merlina mitogenome.

3.5. Phylogenetic relationships

Phylogenetic analyses were conducted using concatenated nucleotide sequences of 13 PCGs and 2 rRNAs obtained from 64 known Satyrinae mitogenomes. P. nepenthes (Charaxinae) and C. davidis (Calinaginae) were used as outgroups (Table 1). Both the ML and BI methods yielded identical topologies in terms of tribal‐level relationships (Figures 6 and 7). 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).

FIGURE 6.

FIGURE 6

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Phylogenetic tree inferred by Maximum likelihood method based on a concatenated matrix of 13 PCGs and 2 rRNAs. Bootstrap support values (BS) are shown at relevant branches.

FIGURE 7.

FIGURE 7

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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.

4. DISCUSSION

In this study, we reported the complete mitogenome of A. merlina for the first time. The newly sequenced mitogenome contained 37 genes and an A + T‐rich region, with the same gene content and arrangement as other published Satyrinae mitogenomes (e.g., Chen et al., 2020; Sun et al., 2021; Yang et al., 2020). The length of the A. merlina mitogenome (15,259 bp) was equal to that of Lethe dura and O. buddha (Dan et al., 2021). Meanwhile, this value fell within the range of the size for other Satyrinae mitogenomes, from 15,122 bp of Melanitis leda (Shi et al., 2013) to 16,129 bp of Bicyclus anynana. The nucleotide composition of the A. merlina mitogenome was rich in A + T (79.9%), with slightly negative AT‐skew (−0.031) and moderately negative GC‐skew (−0.224). Similar trends have been observed in other Satyrinae species, with AT‐skew ranging from −0.055 in Neope muirheadii (Yang et al., 2020) to −0.016 in Hipparchia autonoe (Dan et al., 2021) and the GC‐skew ranging from −0.271 in Stichophthalma louisa to −0.153 in Callerebia polyphemus (Figure 8). Overall, negative AT skews and GC skews are common in Satyrinae. 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., 2018, 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). The A + T‐rich region displayed several structural characteristics commonly 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).

FIGURE 8.

FIGURE 8

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Scatter plot of AT and GC skews in the Satyrinae species.

To better understand their evolutionary relationships, we conducted a preliminary investigation using mitogenomic data. The phylogenetic relationships were reconstructed based on the concatenated nucleotide sequences of 13 PCGs and 2 rRNAs among five tribes (Satyrini, Melanitini, Amathusiini, Elymniini, and Zetherini) and nine subtribes (Lethina, Mycalesina, Parargina, Coenonymphina, Ypthima, Satyrina, Melanargiina, Maniolina, and Erebiina) within Satyrinae.

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.

Satyrini is the most species‐rich tribe in the Satyrinae, comprising approximately 2200 species in 13 subtribes (Marín et al., 2011). 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 monophyletic group, and the genus Lethe was classified as an independent group in our phylogenetic analyses. Correspondingly, Neope and Ninguta were not placed as sister groups to Lethe, which contradicts previous findings (Chen et al., 2020; Dan et al., 2021; Peña et al., 2006). Furthermore, the placement of genus Ninguta varied across our ML and BI trees. It is important to note that this study only analyzed nine out of the 13 subtribes, which limits our ability to fully understand the phylogeny of Satyrini. Therefore, more comprehensive studies with increased sampling of mitogenome sequences are needed to improve our understanding of the evolutionary status and phylogenetic relationships of the entire Satyrini.

5. CONCLUSIONS

The complete mitogenome of an endemic species in China, A. merlina was determined and analyzed in this study. Phylogenetic trees were reconstructed using concatenated nucleotide sequences of 13 PCGs and 2 rRNAs from A. merlina and 63 other known Satyrinae mitogenomes. The ML and BI methods were employed, with P. nepenthes (Charaxinae) and C. davidis (Calinaginae) serving as outgroups. The gene organization and arrangement of the newly sequenced mitogenome were similar to those of other known Satyrinae mitogenomes. Both ML and BI methods produced similar topologies, supporting well‐defined 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 O. buddha within the Satyrina. These results provide valuable information for future studies on the phylogenetic relationships of Satyrinae in future studies.

AUTHOR CONTRIBUTIONS

Qinghui Shi: Conceptualization (equal); data curation (equal); funding acquisition (lead); methodology (equal); project administration (lead); software (equal); validation (equal); writing – original draft (equal); writing – review and editing (lead). Jinling Xie: Conceptualization (equal); data curation (equal); methodology (equal); software (equal); writing – original draft (equal). Jialing Wu: Conceptualization (equal); data curation (equal); methodology (equal); writing – review and editing (equal). Shengchung Chen: Methodology (equal); validation (equal); writing – review and editing (equal). Gang Sun: Methodology (equal); validation (equal); writing – review and editing (equal). Juncheng Zhang: Supervision (lead); validation (equal); writing – review and editing (supporting).

FUNDING INFORMATION

Natural Science Foundation of Fujian Province, Grant/Award Number: 2021J011121; Provincial Training Program of Innovation and Entrepreneurship for Undergraduates, Grant/Award Number: S202311311075.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This work was supported by the Natural Science Foundation of Fujian Province (Grant no. 2021J011121) and the Provincial Training Program of Innovation and Entrepreneurship for Undergraduates (Grant no. S202311311075).

Shi, Q. , Xie, J. , Wu, J. , Chen, S. , Sun, G. , & Zhang, J. (2024). Characterization of the complete mitochondrial genome of an endemic species in China, Aulocera merlina (Lepidoptera: Nymphalidae: Satyrinae) and phylogenetic analysis within Satyrinae. Ecology and Evolution, 14, e11355. 10.1002/ece3.11355

DATA AVAILABILITY STATEMENT

The mitogenome sequence of Aulocera merlina has been deposited on GenBank, and assigned accession number (NC_068667) is provided.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The mitogenome sequence of Aulocera merlina has been deposited on GenBank, and assigned accession number (NC_068667) is provided.

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