The first mitogenomes of the subfamily Epipleminae (Lepidoptera, Uraniidae) and phylogenetic analysis of Macroheterocera

343 The first mitogenomes of the subfamily Epipleminae (Lepidoptera, Uraniidae) and phylogenetic analysis of Macroheterocera Yanpeng Cai1, Aihui Yin1 1 Molecular Diagnostic Research Center, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou, China Corresponding author: Aihui Yin (k[email protected]) Copyright: © Yanpeng Cai & Aihui Yin This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The subfamily Epipleminae, the largest group within Uraniidae (Lepidoptera, Macroheterocera, Geometroidea), comprises small, nocturnal moths primarily distributed in tropical regions. Taxonomic and molecular phylogenetic studies of Epipleminae have long been challenging, and this group remains genetically understudied, with no mitochondrial genomes (mitogenomes) reported to date, despite their wide utility in phylogenetic research. Here, we sequenced, assembled, and annotated the first complete mitogenomes of four epiplemine species: Dysaethria flavistriga (15,404 bp), Monobolodes prunaria (15,258 bp), Phazaca alikangensis (15,482 bp), and Warreniplema fumicosta (15,467 bp), using high-throughput sequencing technology. These mitogenomes exhibit typical gene arrangement of ditrysian Lepidoptera, along with distinctive features such as rare (TA)n microsatellite repeats in the 16S rRNA. Most protein-coding genes (PCGs) initiate with standard ATN start codons and terminate with TAA or a single T residue. Codon usage analysis revealed UUA (Leu2), AUU (Ile), UUU (Phe), AUA (Met), and AAU (Asn) as the five most frequently used codons. All tRNAs display canonical cloverleaf secondary structures, except for trnS1, which lacks the DHU arm. Comprehensive phylogenetic analyses that incorporated existing macroheteroceran mitogenomic data provided robust support for the placement of Epipleminae under Uraniidae, offered the first mitogenome-based evidence supporting the monophyly of Geometroidea on family level, and strongly supported a sister relationship between Geometroidea and Noctuoidea. Key words: 16S rRNA, Epipleminae, Macroheterocera, mitogenome, phylogeny, Uraniidae Introduction The insect mitochondrial genome (mitogenome) is a double-stranded closed loop DNA molecule, measuring 15–18 kb in size. It encodes a pack of 37 genes, including 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes, which exhibit strong conservation across bilaterian metazoans (Boore 1999; Cameron 2014). In addition to the genes, the mitogenome typically contains at least one AT-rich noncoding control region of variable lengths, which serves critical regulatory functions in transcription and replication processes (Clayton 1992; Cameron 2014). The mitogenome’s compact size and high copy number render it significantly easier for lab extraction compared with the nuclear genome (Cameron 2014; Zhang et al. 2023). In recent years, driven Academic editor: Axel Hausmann Received: 10 July 2025 Accepted: 12 September 2025 Published: 15 October 2025 ZooBank: https://zoobank.org/ E8712B0C-B242-4272-AB74C00DF4504AFF Citation: Cai Y, Yin A (2025) The first mitogenomes of the subfamily Epipleminae (Lepidoptera, Uraniidae) and phylogenetic analysis of Macroheterocera. ZooKeys 1255: 343–363. https://doi.org/10.3897/ zookeys.1255.164711 ZooKeys 1255: 343–363 (2025) DOI: 10.3897/zookeys.1255.164711 344 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera by advancements in next-generation sequencing (NGS) technology, insect mitogenome sequence data have indeed been rapidly accumulated and are now widely applied in evolutionary studies, particularly phylogenetics (Curole and Kocher 1999; Cameron 2014; Yang et al. 2019a). The mega-diverse Macroheterocera represents the high-level phylogenetic lineage of the “large moths” in Lepidoptera. It currently includes six superfamilies (Bombycoidea, Drepanoidea, Geometroidea, Lasiocampoidea, Mimallonoidea, and Noctuoidea), and consists of more than 72,400 described species (van Nieukerken et al. 2011; Mitter et al. 2017; Rota et al. 2022). A substantial number of molecular phylogenetic studies involving Macroheterocera have been carried out in the recent two decades (e.g. Mutanen et al. 2010; Bazinet et al. 2013; Heikkilä et al. 2015; Kawahara et al. 2019; Cheng et al. 2022). It is shown that Bombycoidea, Lasiocampoidea, Geometroidea and Noctuoidea are no doubt closely related within Macroheterocera, although their interrelationships are still debatable (Cheng et al. 2022; Rota et al. 2022). Mimallonoidea and Drepanoidea, on the other hand, are highly labile regarding their phylogenetic affinities. In most previous studies, they are recovered and accepted as two basal lineages that successively branched off from the remaining macroheterocerans (Bazinet et al. 2013; Timmermans et al. 2014; Kawahara et al. 2019; Yang et al. 2019b; Mayer et al. 2021; Rota et al. 2022). Yet in some studies, Mimallonoidea has been found to form a subclade within Drepanoidea at the base of Macroheterocera (Regier et al. 2009; Regier et al. 2013), or group together with Bombycoidea + Lasiocampoidea (Wang et al. 2019; Chen et al. 2022), or even be placed far apart from other macroheterocerans (Mutanen et al. 2010), while Drepanoidea’s placement within Macroheterocera is even more unstable, with its phylogenetic position conflicting across analyses (Mutanen et al. 2010; Heikkilä et al. 2015; Wang et al. 2019; Cheng et al. 2022; Zheng et al. 2022). Uraniidae is a family within the superfamily Geometroidea of Macroheterocera, and comprises ~700 species in ~90 genera (Minet and Scoble 1998; van Nieukerken et al. 2011). This family is distributed worldwide, with its members predominantly found in the pantropical regions (Holloway 1998). Uraniids differ from other Lepidopterans by having unique sexually dimorphic tympanal organs (Scoble 1992). They are delicate, small to large, broad-winged moths; their hindwing termens are often angulate or with tail-shaped projections (Minet and Scoble 1998). Some swallowtail-like diurnal species are renowned for their brilliant iridescent coloration and prominently tailed hindwings (Scoble 1992). Epipleminae, the most speciose subfamily of the four subfamilies (the other three: Auzeinae, Microniinae, and Uraniinae) in Uraniidae (Minet and Scoble 1998), was established by Hampson (1892) originally as a family based on very simple diagnostic characters of the wing venation. Modern phylogenetic revisions have redefined its status as a subfamily under Uraniidae by the synapomorphic character of the tympanal organ (Sick 1937; Minet 1983, 1986). It currently includes over 600 species in ~70 genera (Sohn and Yen 2005), and is primarily distributed in pantropical montane zones (Holloway 1998; Smetacek 2005). Compared with the other three subfamilies, the epiplemines are generally small, drab-colored nocturnal moths (Scoble 1992; Minet and Scoble 1998). The resting posture is a distinctive feature of this subfamily (Sohn and Yen 2005). Typically, their hindwings are folded along the abdomen, while forewings are extended horizontally, either flat or longitudinally rolled, the entire 345 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera body forms a T-shaped dorsal profile when viewed from above (Minet and Scoble 1998; Sohn and Yen 2005). Like many early-established lepidopteran taxa, the generic taxonomy of Epipleminae has remained highly unsatisfactory since its establishment, and a robust phylogenetic framework for this subfamily is urgently needed as well (Holloway 1998; Sohn and Yen 2005). Unfortunately, no molecular phylogenetic study focusing on Epipleminae or Uraniidae has been published to date. Despite the significant utility of mitogenomes in phylogenetic research, only three mitochondrial genome sequences have been published for Uraniidae (as of 8 July 2025), covering just two species from two subfamilies, Microniinae and Uraniinae. Notably, no mitogenome sequences are available for Epipleminae, the largest subfamily within Uraniidae. Accordingly, in this study, we present four complete mitogenome sequences for Epipleminae for the first time. Our focal species include Dysaethria flavistriga, Monobolodes prunaria, Phazaca alikangensis, and Warreniplema fumicosta, of which the specimens were all collected from Guizhou, China. We provide a detailed description and comparative analysis of these mitogenomes. Additionally, we explore the phylogenetic positions of Epipleminae and Uraniidae, and further reconstruct the phylogeny of Macroheterocera using available mitogenome data. Materials and methods Sample collection and identification Adult specimens of the four epiplemine species were captured by light trap from Baiyan Village (25°21'47"N, 107°56'5"E, 748 m elevation), Maolan National Nature Reserve, Libo County, Guizhou, China on 8 July 2024. The specimens were pinned and dried, and their genitalia were dissected and slide-mounted for examination. Identification was based on external morphology and genitalia characters, following taxonomic references (Holloway 1998; Sohn and Yen 2005; Choi et al. 2024). DNA extraction and sequencing Total DNA was extracted from the thoracic muscles of the dry specimens using the MagicMag Genomic DNA Micro Kit (Sangon Biotech Co., Shanghai, China) following the manufacturer’s protocol. For each sample, 0.2 μg DNA was fragmented by sonication to an average size of 350 bp. The fragmented DNA was then subjected to high-throughput pair-ended sequencing (PE150) on Illumina NovaSeq 6000 platform at Novogene Bioinformatics Technology Co., Ltd (Tianjin, China). Mitogenome assembly, annotation, and analysis The raw data were processed using Fastp v. 0.19.7 (Chen et al. 2018) to remove low-quality reads, yielding ~3 Gb of clean data for each sample. The complete mitogenomes were assembled de novo using MitoZ v.3.6 (Meng et al. 2019) and SPAdes v. 4.0.0 (Prjibelski et al. 2020). The assembled sequences were polished with Pilon v. 1.24 (Walker et al. 2014), and annotated using MitoZ software and MITOS2 online service (Galaxy v. 2.1.9) (Al Arab et al. 2017; Donath et 346 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera al. 2019), followed by manual verification. Final mitogenome maps were generated using Organellar Genome DRAW (OGDRAW) (Lohse et al. 2013). The mitogenomes were then uploaded to GenBank. The standard 658 bp COX1 barcodes were extracted from the mitogenome sequences and uploaded to the BOLD system. The corresponding GenBank accession numbers and BOLD sample IDs are listed in Table 1. The base composition of the mitogenomes was analyzed using MEGA v. 11.0.13 (Tamura et al. 2021). Nucleotide composition bias between the two strands was calculated using the formulas proposed by Perna and Kocher (1995): AT-skew = (A-T)/(A+T) and GC-skew = (G-C)/(G+C). Relative synonymous codon usage (RSCU) diagrams for the PCGs were generated and analyzed using PhyloSuite v. 1.2.3 (Zhang et al. 2020; Xiang et al. 2023). The non-synonymous substitution rate (Ka), synonymous substitution rate (Ks), and their ratio (Ka/Ks) for each PCG in Epipleminae were calculated through DnaSP v. 5.10.01 (Librado and Rozas 2009). The secondary structures of the tRNAs were predicted via MITOS2 online service, while rRNA secondary structures were predicted with R2DT v. 1.3 (Sweeney et al. 2021), both with manual verification. Tandem repeat elements in the control regions were identified using Tandem Repeats Finder (https:// tandem.bu.edu/trf/home) (Benson 1999). Phylogenetic analyses To determine the phylogenetic positions of Epipleminae and Uraniidae, and to reconstruct the phylogeny of Macroheterocera, we aimed to maximize taxonomic representation at the family and subfamily level. We downloaded mitogenomic sequences for 34 species from GenBank, representing 18 families within Macroheterocera and four outgroup families (Table 2). In addition, we assembled four novel mitogenomic sequences for Epipleminae in this study. For three other families (Cimeliidae, Pseudobistonidae, and Sematuridae) and one subfamily (Cyclidiinae of Drepanidae), which lack publicly available mitogenomic data but have accessible genomic/transcriptomic SRA files (NCBI), we assembled mitogenomic sequences of five species (Table 2). However, due to the low quality of the SRA data, the three sequences for Cimeliidae, Pseudobistonidae, and Cyclidiinae were only partially assembled. In our phylogenetic analyses, multi-gene and multi-method approaches were employed to mitigate single-locus bias, resolve topological conflicts, and strengthen nodal support, thereby ensuring robust phylogenetic hypotheses. Consequently, a total of ten phylogenetic trees were reconstructed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods based on five data Table 1. The GenBank accession numbers and BOLD sample IDs of the four epiplemine species. Species Accession numbers (GenBank) Sample IDs (BOLD) Dysaethria flavistriga PV151521 GZUTCM:SWD Monobolodes prunaria PV151522 GZUTCM:SWM Phazaca alikangensis PV151523 GZUTCM:SWP Warreniplema fumicosta PV151524 GZUTCM:SWW 347 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera Table 2. Basic information of the mitogenomes used for the phylogenetic analyses in this study. Accession numbers of newly sequenced species are highlighted in bold. Superfamily Family Subfamily Species Accession/SRA number Ingroup Bombycoidea Bombycidae Bombycinae Bombyx mandarina AB070263 Brahmaeidae – Brahmaea hearseyi KU884326 Endromidae – Andraca theae KX365419 Eupterotidae – Ganisa cyanogrisea MF100140 Saturniidae Saturniinae Antheraea pernyi MK920216 Sphingidae Macroglossinae Theretra clotho MZ562564 Drepanoidea Cimeliidae – Axia margarita SRR1006157 Doidae – Doa sp. KJ508058 Drepanidae Cyclidiinae Cyclidia substigmaria SRR1021608 Drepaninae Pseudalbara parvula MZ823341 Oretinae Oreta sp. MZ823343 Thyatirinae Tethea albicostata OK149234 Geometroidea Epicopeiidae – Epicopeia hainesii MK033610 Geometridae Alsophilinae Alsophila aescularia OX276387 Ennominae Ectropis grisescens MN792921 Geometrinae Iotaphora admirabilis MK903032 Larentiinae Hydrelia parvulata MN962739 Sterrhinae Idaea salutaria MK122626 Pseudobistonidae Pseudobistoninae Pseudobiston pinratanai SRR11994912, SRR13187626 Sematuridae – Homidiana leachi SRR27474561 –Mania lunus SRR27474554, SRR27474556 Uraniidae Epipleminae Dysaethria flavistriga PV151521 Monobolodes prunaria PV151522 Phazaca alikangensis PV151523 Warreniplema fumicosta PV151524 Microniinae Acropteris iphiata MN093120 Uraniinae Lyssa zampa MZ713634 Lasiocampoidea Lasiocampidae Chaetomalachiinae Euthrix laeta KU870700 Pinarinae Trabala vishnou MZ927091 Mimallonoidea Mimallonidae – Lacosoma valva KJ508050 Noctuoidea Erebidae Arctiinae Brunia dorsalis MN635735 Erebinae Erebus caprimulgus MZ959074 Lymantriinae Dasychira tristis MZ520324 Euteliidae Euteliinae Eutelia adulatricoides KJ185131 Noctuidae Amphipyrinae Spodoptera frugiperda KU877172 Dilobinae Diloba caeruleocephala OX381635 Eustrotiinae Maliattha signifera OQ111926 Nolidae Eariadinae Earias clorana OK235312 Notodontidae Dudusinae Dudusa sphingiformis MW788876 Outgroup Calliduloidea Callidulidae – Pterodecta felderi MT370823 Hyblaeoidea Hyblaeidae – Hyblaea puera MW885970 Papilionoidea Papilionidae Parnassiinae Luehdorfia chinensis KM453725 Pyraloidea Pyralidae Phycitinae Plodia interpunctella KT428892 348 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera matrices: (1) “PCG123R” matrix: All three codon positions of 13 PCGs plus two rRNA genes (13,214 sites); (2) “PCG123” matrix: All three codon positions of 13 PCGs (11,163 sites); (3) “PCG12R” matrix: The 1st and 2nd codon positions of PCGs plus two rRNA genes (9,493 sites); (4) “PCG12” matrix: The 1st and 2nd codon positions of PCGs (7,442 sites); (5) “AA” matrix: Amino acid sequences translated from 13 PCGs (3,721 sites). The DNA sequences of each gene were aligned separately using MAFFT v. 7.52 (Katoh and Standley 2013) with the L-INS-i algorithm. Poorly aligned positions were removed by trimAI v. 1.4.1 (Capella-Gutiérrez et al. 2009). Individual genes were then concatenated in MEGA 11 software to generate the five datasets mentioned above. Compositional heterogeneity among sequences was analyzed separately for each dataset with AliGROOVE v. 1.0.8 (Kück et al. 2014). For the AA matrix, the site-heterogeneous substitution model (CAT + GTR) was used in the BI analysis. This model was chosen because it demonstrated better performance against systematic errors and long-branch attraction (LBA) artifacts compared to site-homogeneous models, despite its higher computational demand. For all other tree-building analyses, site-homogeneous models were applied, and the optimal partitioning schemes and corresponding best-fit substitution models were determined using ModelFinder v. 2.2.2 (Kalyaanamoorthy et al. 2017) implemented in IQ-TREE2 v. 2.3.6 (Minh et al. 2020) (Suppl. material 2: tables S1–S9). The ML trees were constructed in IQ-TREE2 software with 10,000 ultrafast bootstraps (BS) (Hoang et al. 2018). The BI tree from AA matrix was generated via PhyloBayes-MPI v. 1.8c (Lartillot et al. 2013), with two independent searches of 50,000 generations until convergence (maxdiff < 0.1). The BI trees for the other four datasets were generated via MrBayes v. 3.2.7 (Ronquist et al. 2012), with four independent chains run for 5–7 million generations until the average standard deviation of split frequency dropped below 0.01. In each BI analysis, the initial 25% of trees were discarded as burn-in, and consensus trees with posterior probabilities (PP) for each node were computed from the remaining trees. All phylogenetic trees were visualized in FigTree v. 1.4.4 (Rambaut et al. 2018). Results and discussion Mitogenome structure and organization The complete mitogenomes of D. flavistriga (length: 15,404 bp), M. prunaria (length: 15,258 bp), P. alikangensis (length: 15,482 bp), and W. fumicosta (length: 15,467 bp) were sequenced and annotated. As expected, these epiplemine species shared the typical composition of 37 genes (13 PCGs, 22 tRNA genes, and two rRNA genes) and one A+T-rich control region with other metazoan animals (Wolstenholme 1992) (Fig. 1). Their gene arrangement pattern followed that of most other ditrysian moths (Cao et al. 2012; Park et al. 2016), with nine PCGs and 14 tRNAs encoded on the majority (J) strand, while the remaining four PCGs, eight tRNAs and the two rRNAs on the minority (N) strand (Suppl. material 2: table S10). The mitogenomes of the four epiplemine species exhibited a distinct overall A + T nucleotide bias (80.8% to 81.2%). This high A+T content varied slightly among different types of genes, being most pronounced in the control region 349 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera ND5 COX1 ND4 CYTB ND2 ND1 COX3 COX2 AT P6 ND6 ND3 ND4L AT P8 tRNA-Leu tRNA-Lys tRNA-Gln tRNA-Cys tRNA-Met tRNA-Phe tRNA-Gly tRNA-Trp tRNA-Asp tRNA-Leu tRNA-Ser tRNA-Ile tRNA-Tyr tRNA-His tRNA-Glu tRNA-Asn tRNA-Val tRNA-Ala tRNA-Thr tRNA-Ser tRNA-Pro tRNA-Arg Dysaethria flavistriga mitochondrial genome 15,404 bp 12S ribosomal RNA controlregion 16S ribosomal RNA ND5 COX1 ND4 CYTB ND2 ND1 COX3 COX2 AT P6 ND6 ND3 ND4L AT P8 tRNA-Lys tRNA-Leu tRNA-Gln tRNA-Cys tRNA-Glu tRNA-Met tRNA-Leu tRNA-Thr tRNA-Val tRNA-Asn tRNA-Gly tRNA-Asp t RNA-Trp tRNA-Phe tRNA-Ala tRNA-Ser tRNA-His tRNA-Ile tRNA-Tyr tRNA-Pro t RNA-Ser tRNA-Arg Monobolodes prunaria mitochondrial genome 15,258 bp 16S ribosomal RNA 12S ribosomal RNA controlregion 16S ribosomal RNA ND5 COX1 ND4 CYTB ND2 ND1 COX3 COX2 AT P6 ND6 ND3 ND4L AT P8 tRNA-Lys tRNA-Gln tRNA-Leu tRNA-Met tRNA-Cys tRNA-Val tRNA-Thr tRNA-Phe tRNA-Glu tRNA-Leu tRNA-Gly tRNA-Ser tRNA-Asp tRNA-Tyr tRNA-Trp tRNA-His tRNA-Asn tRNA-Ile tRNA-Ala tRNA-Pro tRNA-Arg tRNA-Ser Phazaca alikangensis mitochondrial genome 15,482 bp control region 12S ribosomal RNA ND5 COX1 16S ribosomal RNA ND4 CYTB ND2 ND1 COX3 12S ribosomal RNA COX2 AT P6 ND6 controlregion ND3 ND4L AT P8 tRNA-Leu tRNA-Lys tRNA-Gln tRNA-Cys tRNA-Met tRNA-Asp tRNA-Gly tRNA-Leu tRNA-Thr tRNA-Glu tRNA-Trp tRNA-Phe tRNA-Asn tRNA-Ile tRNA-His tRNA-Ala tRNA-Tyr tRNA-Ser t RNA-Val tRNA-Pro tRNA-Arg tRNA-Ser W arreniplema fumicosta mitochondrial genome 15,467 bp control region ribosomal RNAs transfer RNAs cytochrome b ATPsynthase complex IV (cytochrome c oxidase) complex I (NADH dehydrogenase) Figure 1. Circular structures of the mitochondrial genomes of four newly sequenced epiplemine species. Genes are represented with different color blocks. Colored blocks outside of each ring indicate that the genes are on the majority strand, while colored blocks within the rings indicate that the genes are located on the minority strand. (93.7% to 95.6%), followed by rRNAs (84.9% to 85.7%), tRNAs (81.5% to 82.1%), and lowest in PCGs (79.0% to 79.8%) (Table 3). The AT-skews of the mitogenomes were not significant either across the entire sequences or in different coding regions (-0.085 to 0.044) (Table 3). In contrast, the GC-skew values were much greater and consistently negative, both across the whole mitogenomes and in different coding regions (-0.667 to -0.091) (Table 3). Gene overlaps and intergenic spacers were identified in the mitogenomes of the four epiplemine species: D. flavistriga (6 overlaps, 15 spacers), M. prunaria (5 overlaps, 18 spacers), P. alikangensis (8 overlaps, 15 spacers), and W. 350 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera fumicosta (8 overlaps, 14 spacers) (Suppl. material 2: table S10). The gene overlaps ranged in size from 1 to 8 bp, with the longest overlap (8 bp, sequence: AAGCCTTA) occurring between the trnW and trnC genes in all epiplemine species (Suppl. material 2: table S10). The intergenic spacers ranged in size from 1 to 100 bp, with the longest spacer located between the trnQ and ND2 genes, measuring 56 bp in D. flavistriga, 54 bp in M. prunaria, 100 bp in P. alikangensis, and 57 bp in W. fumicosta (Suppl. material 2: table S10). A long noncoding spacer between the trnQ and ND2 genes appears to be a common feature among all lepidopterans. PCGs and codon usage The total lengths of the 13 PCGs in the mitogenomes of D. flavistriga, M. prunaria, P. alikangensis and W. fumicosta were 11,200 bp, 11,200 bp, 11,202 and 11,199 bp respectively. Most PCGs in the four mitogenomes used the typical ATN start codon for initiation (Suppl. material 2: table S10). However, several genes exhibited unconventional start codons: The putative codon CGA was used in COX1 in all four species; GTG was used in ATP6 in D. flavistriga; and TTG was used in ND1 in M. prunaria, P. alikangensis and W. fumicosta (Suppl. material 2: table S10). Regarding stop codons, most PCGs in the four mitogenomes terminated with either the standard stop codon TAA (observed in ND2, ATP8, ATP6, COX3, ND3, ND4L, ND6, and CYTB in all four species; ND5 in three species; ND4 and ND1 in one species), or a single T residue (observed in COX1 and COX2 in all four species; ND4 in three species; ND1 in two species; ND5 in one species) (Suppl. material 2: table S10). The only exception was ND1 gene in W. fumicosta, which terminated with TAG (Suppl. material 2: table S10). The relative synonymous codon usages (RSCU) for the four mitogenomes are shown in Fig. 2. The analysis revealed that the five most frequently used codons were UUA (Leu2), AUU (Ile), UUU (Phe), AUA (Met), and AAU (Asn). In contrast, several codons were absent in these mitogenomes: ACG (Thr), AGG (Ser1), and CUC (Leu1) in D. flavistriga; ACG (Thr), AGC (Ser1), AGG (Ser1), CCG (Pro), CGC (Arg), GGC (Gly), and UGG (Trp) in M. prunaria; AGG (Ser1) and CGC (Arg) in P. alikangensis; AGG (Ser1), CUC (Leu1), CUG (Leu1), GUC (Val) and UCG (Ser2) in W. fumicosta. AGG (Ser1) was the only codon absent in all four species. In conclusion, synonymous codons ending with A or T were strongly preferred over those ending with C or G when encoding the same amino acid. Table 3. Base composition in different regions in the mitochondrial genomes of four epiplemine species: D. flavistriga/M. prunaria/P. alikangensis/W. fumicosta. Feature Size (bp) A+T% AT-skew GC-skew Whole mitogenome 15404/15258/ 15482/15467 81.2%/81.2%/ 80.8%/81.2% 0.007/-0.0004/ 0.020/0.021 -0.187/-0.172/ -0.200/-0.195 PCGs 11200/11200/ 11202/11199 79.7%/79.8%/ 79.0%/79.4% 0.009/0.003/ 0.018/0.019 -0.170/-0.152/ -0.181/-0.176 rRNA genes 2177/2179/ 2195/2237 85.2%/85.4%/ 84.9%/85.7% 0.009/-0.009/ 0.015/0.027 -0.344/-0.342/ -0.372/-0.352 tRNA genes 1467/1459/ 1461/1458 82.0%/82.0%/ 82.1%/81.5% 0.024/0.038/ 0.044/0.034 -0.091/-0.125/ -0.115/-0.122 Control region 431/226/371/411 93.7%/93.8%/ 95.4%/95.6% -0.059/-0.085/ -0.028/0.038 -0.630/-0.143/ -0.412/-0.667 351 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera Gl nHis Asn Pro Thr Leu1 Gl uMet Ar gTyr As pLys Ala Ile Ser1 Ser2 Leu2 Cy sTrp Va lGly Phe GCU UGUGAUGAA UUU GGUCAU AUU AAA UUA CUU AUAAAU CCUCAA CGU UCUAGUACU GUUUGA UAU GCC UGCGACGAG UUCGGCCAC AUCAAG UUGCUC AUGAAC CCCCAG CGC UCCAGCACC GUCUGGUAC GCA GGACUACCA CGA UCAAGAACA GUA GCG GGG CUGCCG CGG UCGAGGACG GUG Gl nHis Asn Pro Thr Leu1 Gl uMet Ar gTyr As pLys Ala Ile Ser1 Ser2 Leu2 Cy sTrp Va lGly Phe 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 RSCURSCU RSCU RSCU Dysaethria flavistriga Monobolodes prunaria Phazaca alikangensis Warreniplemа fumicostа GCU UGUGAUGAA UUU GGUCAU AUU AAA UUA CUU AUAAAU CCUCAA CGU UCUAGUACU GUUUGA UAU GCC UGCGACGAG UUCGGCCAC AUCAAG UUGCUC AUGAAC CCCCAG CGC UCCAGCACC GUCUGGUAC GCA GGACUACCA CGA UCAAGAACA GUA GCG GGG CUGCCG CGG UCGAGGACG GUG Figure 2. Relative synonymous codon usages (RSCU) of four newly sequenced epiplemine species. Codon families are indicated below the X-axes. The synonymous substitution rates (Ks), non-synonymous substitution rates (Ka), and the Ka/Ks ratios of the 13 PCGs in Epipleminae were calculated (Fig. 3). The Ka/Ks values were used to estimate the natural selective pressures acting on the PCGs within the population (Hurst 2002). In this study, the Ka/Ks values for all 13 PCGs were less than one (0.02 in COX1 to 0.40 in ATP8), indicating varying levels of purifying selection across these genes (weakest in COX1 and strongest in ATP8). tRNA genes, rRNA genes, and the control region The total lengths of the 22 tRNA genes in the mitogenomes of D. flavistriga, M. prunaria, P. alikangensis and W. fumicosta were 1,467 bp, 1,459 bp, 1,461 and 1,458 bp, respectively. The lengths of individual tRNA genes ranged from 62 bp (the shortest, trnS1, in P. alikangensis and W. fumicosta) to73 bp (the longest, trnL1, in D. flavistriga) (Suppl. material 2: table S10). The predicted secondary structures of these tRNAs are shown in Suppl. material 1: figs S1–S4. All 22 tRNAs folded into a typical cloverleaf secondary structure, except for trnS1, which lacked the dihydrouridine (DHU) arm, and instead possessed a single-stranded loop. Wobbled G-U pairs were observed in many tRNA secondary structures; however, they were absent in trnD, trnE, trnH, trnM, trnR, trnS2, and trnY in all four species. Additionally, the rarer U-U pairing was detected, but only in trnA, trnL2 and trnS2 in all four species. The total lengths of the two rRNA genes in the mitogenomes of D. flavistriga, M. prunaria, P. alikangensis and W. fumicosta were 2,177 bp, 2,179 bp, 2,195 and 2,237 bp respectively. The 16S rRNA genes were located between trnL1 and trnV, and the 12S rRNA genes were positioned between trnV and the control region, with no intergenic spacers or overlaps observed (Suppl. material 2: table S10). The 16S rRNA gene lengths ranged from 1,394 bp (in D. flavistriga) to 1,458 bp (in W. fumicosta), nearly twice the length of the 12S rRNA gene, which ranged from 779 bp (in M. prunaria and W. fumicosta) to 783 bp (in D. flavistriga and P. alikangensis) (Suppl. material 2: table S10). The predicted secondary structures of the two rRNAs for all four species are shown in Fig. 4, Suppl. material 1: figs S5–S11. 358 ZooKeys 1255: 343–363 (2025), DOI: 10.3897/zookeys.1255.164711 Yanpeng Cai & Aihui Yin: Mitogenomes of Epipleminae and phylogenetic analysis of Macroheterocera Author ORCIDs Yanpeng Cai https://orcid.org/0000-0001-6554-3784 Aihui Yin https://orcid.org/0000-0001-7614-9969 Data availability Data presented in this study are openly available in the GenBank repository under accession numbers: PV151521–PV151524 and in the BOLD system with sample IDs GZUTCM:SWD, GZUTCM:SWM, GZUTCM:SWP, and GZUTCM:SWW. References Al Arab M, zu Siederdissen CH, Tout K, Sahyoun AH, Stadler PF, Bernt M (2017) Accurate annotation of protein-coding genes in mitochondrial genomes. 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Phylogenetic trees of Macroheterocera. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1255.164711.suppl1 Supplementary material 2 Supplementary tables Authors: Yanpeng Cai, Aihui Yin Data type: pdf Explanation note: tables S1–S9. The corresponding partitioning schemes and substitution models determined by ModelFinder for the phylogenetic analyses using site-homogeneous models. table S10. Annotation of the mitochondrial genomes of four epiplemine species. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1255.164711.suppl2