Skip to main content
NCBI home page
Mitochondrial DNA. Part B, Resources logoLink to Mitochondrial DNA. Part B, Resources
. 2026 Oct 14;11(11):1328–1332. doi: 10.1080/23802359.2026.2742662

The complete mitochondrial genomes of Colias lada and Colias wanda (Lepidoptera: Pieridae: Coliadinae) and their phylogenetic analyses

Wang Sunle a, Gu Xuesong b, Zhu Zhenyao c, Wang Ying b,✉
PMCID: PMC13670270  PMID: 42889812

Abstract

The complete mitochondrial genomes of two Qinghai-Tibet Plateau butterflies, Colias lada and C. wanda, were assembled from next-generation sequencing data, measuring 15,078 bp and 15,144 bp. Both contain 13 PCGs, 22 tRNAs, 2 rRNAs and one control region. Phylogenetic trees based on the 13 PCGs and 2 rRNAs using ML and BI methods, with high support values, show that C. wanda, C. lada and C. sifanica share a recent common ancestor, and reconstruct the relationships among the Coliadinae genera.

Keywords: Colias, mitogenome, phylogenetic inference, evolution

Introduction

Pieridae is one of the major butterfly groups, comprising at least 1,159 species in 86 genera worldwide (Carvalho et al. 2024). The group exhibits variations in wing coloration, morphology, and ecological traits, serving as a model for research on community ecology, speciation, biogeography, and interactions (Wahlberg et al. 2014). Although the monophyly of the four subfamilies in Pieridae is well established (Nie et al. 2018; Wei et al. 2022), the phylogenetic relationships within the rapidly diverging Colias in Coliadinae still remain unstable (Wheat et al. 2006; Carvalho et al. 2024, Mo et al. 2025).

The typical lepidoptera mitochondrial genome is a circular and compact molecule that is 14 to 16 kb in size and composed of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and a noncoding region (control region). It has been widely used in studies of taxonomic identification, phylogeny, and molecular evolution (Hao et al. 2014; Wahlberg et al. 2014; Cao et al. 2016; Yong et al. 2017; Wei et al. 2022). To date, about 66 species have been reported by Mo et al. (2025); however, the complete mitogenome sequences for both C. lada and C. wanda are not yet available. Morphologically, both C. lada and C. wanda exhibit predominantly orange-yellow wings with dark marginal bands, discal spots, and yellowish-white or orange markings. The two species can be distinguished by wing patterns: C. lada has narrower, toothed marginal bands and brighter wings, whereas C. wanda displays wider marginal bands, distinct black basal streaks near the wing base, and overall darker wings (Wu and Xu 2017). Here, we sequenced and characterized the mitogenomes of C. lada and C. wanda, two species with similar morphological characteristics that mostly inhabit high-altitude regions. These data are valuable for resolving robust phylogenetic relationships and investigating adaptive evolution in this butterfly genus, providing insights into potential mechanisms of high-altitude adaptation.

Materials and methods

Adult C. wanda and C. lada samples were collected from Huashixia Town, Qinghai, China (N 35.10167°, E 98.86083°, 4,310 m above sea level) in July 2020 and Zheduo Mountain, Kangding, Sichuan, China (N 30.1807°, E 101.7518°, 4,369 m above sea level) in June 2022, respectively (Figure 1). The samples were preserved in absolute ethanol in the field and subsequently stored at −80 °C at the College of Life Sciences, Anhui Normal University (www.ahnu.edu.cn; Chengyong Su, sucy@ahnu.edu.cn) under the voucher numbers CW_HSX and CL_ZD.

Figure 1.

Dorsal and ventral views of two butterfly species. Species A has bright orange dorsal and greenish-yellow ventral. Species B features broader brown margins on the dorsal and dark hindwing spots on the ventral, illustrating color differences.

Open in a new tab

Species reference images of colias lada and colias wanda. (A) C. lada. The wings are pale orange-yellow; the forewing has a small dark spot at the end of the disk cell on the upper side, and both the forewing and hindwing outer margins have a pale, blackish, broad band. (B) C. wanda, dorsal view. The upper side is orange-yellow, the forewing discal-cell-end spot is black, and the outer-margin black band is well-developed. The hindwing base features a well-developed yellow patch, and the outer margin is more rounded/blunt. The images were taken by Sunle Wang.

Genomic DNA extraction, library construction, and sequencing were performed as described in a previous study (Wei et al. 2022). The mitogenomes were assembled with GetOrganelle v1.7.0 (Jin et al. 2020), annotated using MITOS2 (Donath et al. 2019), and visualized via the CGView Web server (https://cgview.ca). PhyloSuite v1.2.3 (Xiang et al. 2023) was used for RSCU and phylogenetic analyses. Initial alignments of the 13 protein-coding genes (PCGs) were generated using the integrated MAFFT module and subjected to manual inspection. Based on previous phylogenetic studies, we selected Leptidea morsei as the outgroup (Wang et al. 2024). Phylogenetic reconstruction was performed using both IQ-TREE (Nguyen et al. 2015; Minh et al. 2020) and Bayesian inference (Ronquist et al. 2012), with Leptidea morsei designated as the outgroup. Branch support was assessed with 5,000 ultrafast bootstrap (UFBoot) replicates, 10,000 SH-like approximate likelihood ratio tests (SH-aLRT), and approximate Bayesian tests. Bayesian analyses were conducted in MrBayes 3.2.6 (Ronquist et al. 2012), as performed in a previous study (Wang et al. 2024).

Results

The complete mitochondrial genomes of C. lada and C. wanda are 15,078 bp and 15,144 bp in length, respectively (Figure 2). Both genomes contain 13 PCGs, two rRNAs, 22 tRNAs, and one A + T-rich region. The gene order is conserved compared with those of previously sequenced Pieridae species. The overall A + T contents are 81.0% (C. lada) and 81.2% (C. wanda), reflecting a strong A + T bias.

Figure 2.

Circular genome maps of *Colias lada* (15078 bp) and *Colias wanda* (15144 bp), detailing gene organization, GC content, and skew patterns. The figure presents two circular genome maps for *Colias lada* (panel a) and *Colias wanda* (panel b), each depicting mitochondrial genome features. The outer circle showcases colored segments representing gene types: coding sequences (CDS in purple), transfer RNA (tRNA in pink), ribosomal RNA (rRNA in red), and the D-loop (green). The inner circles list genome lengths (15078 bp for *Colias lada*, 15144 bp for *Colias wanda*) and display GC content with positive (green) and negative (purple) skew patterns. Labeled genes such as COI, ND1, and others illustrate their positions along the circular genomes.

Open in a new tab

(a) Gene arrangement in the mitogenome of colias lada; (b) gene arrangement in the mitogenome of colias wanda.

The two newly sequenced mitochondrial genomes exhibit consistent relative synonymous codon usage (RSCU) patterns (Figure S1; Table S1 and Table S2), with all 62 available codons corresponding to 22 amino acids in 13 PCGs of the subfamily Coliadinae. In both species, the preferred codons were UUA, AUU, and UUU. Moreover, the most frequently used amino acids are Leu2, Ile, and Phe, which are thought to be closely linked to the high A + T content of the mitochondrial genome.

Phylogenetic trees based on maximum likelihood and Bayesian inference analyses consistently supported C. lada as the sister species to C. sifanica (Figure 3) and indicated a recent common ancestor for C. wanda, C. lada, and C. sifanica with high support values (IQ-TREE: SH-aLRT = 99.3%, UFBoot = 100%, standard bootstrap = 100%; Bayesian: PP = 1.0). The concordance among the various methods reinforces the credibility of the phylogenetic relationships inferred for Coliadinae in this study.

Figure 3.

Phylogenetic tree illustrating butterfly relationships with species names, branch lengths, and bootstrap values. The figure depicts a phylogenetic tree showing evolutionary relationships among butterfly species in the Lepidoptera order. It is divided into two primary groups: Dismorphane and Coliadinae. Each branch is labeled with numerical values indicating bootstrap support, with examples like "Leptidea morsei" (0.1215, 98.41/97) and "Colias lada" (PZ934426, 99.3/100). The tree spans various taxa from genera including Leptidea, Eurema, and Colias, demonstrating clustering and divergence among species. Branch lengths reflect evolutionary distance, highlighted with a scale bar measuring 0.03.

Open in a new tab

Phylogenetic relationships of coliadinae species based on ML and BI analyses using the nucleotide sequences of 13 PCGs and two rRNA genes. ML analysis was performed under the GTR+I + G model, and BI analysis was conducted using the GTR+I model. Node support values are shown near each node in the order of ML ultrafast bootstrap support (UFBoot), bayesian posterior probability (PP), and the ML SH-like approximate likelihood ratio test (SH-aLRT). The sequences of L. morsei NC022686 (Hao et al. 2014), E. blanda NC032285 (Yong et al. 2017), D. lycorias OP779725 (Wei et al. 2022), G. mahaguru NC026837 (Yang et al. 2016), G. rhamni NC026046 (Wang et al. 2015), C. pomona NC022687 (Hao et al. 2014), C. erate NC027253 (Wu et al. 2016), C. fieldii NC053715 (Xian et al. 2021), and other sequences deposited in the NCBI sequence read archive were assembled by Wang et al. (2024).

Discussion and conclusions

In this study, we sequenced the complete mitochondrial genomes of C. lada and C. wanda for the first time and performed comparative mitochondrial genomic and phylogenetic analyses. The results revealed that their genome structures are highly conserved relative to those of other Pieridae species, with pronounced A + T bias and similar codon usage patterns. Phylogenetic trees reconstructed using both maximum likelihood (ML) and Bayesian inference (BI) produced identical topologies, providing a molecular basis for clarifying the phylogenetic relationships among genera within Coliadinae. The recovered sister relationships among C. lada, C. wanda, and C. sifanica are consistent with their shared distribution across the Qinghai-Tibet Plateau and adjacent high-altitude regions, paralleling the close phylogenetic affinities among montane Colias lineages reported by Mo et al. (2025). This close relationship is further supported by morphological evidence. Both C. lada and C. wanda exhibit predominantly orange-yellow wings with dark marginal bands, discal spots, and yellowish-white or orange markings. They can be distinguished from one another mainly by wing-pattern details: C. lada has narrower, toothed marginal bands and brighter wings, whereas C. wanda displays wider marginal bands, distinct black basal streaks near the wing base, and overall darker wings (Wu and Xu 2017). The short branch lengths separating these three species in Figure 3 reflect their recent and rapid diversification, a pattern also noted for the genus as a whole (Mo et al. 2025). Although Mo et al. (2025) conducted extensive sequencing and systematic sampling of the genus Colias on the basis of large-scale nuclear genomic data, most of the taxa involved in their study currently lack publicly available complete mitochondrial genome data. The taxon sampling in the present study was constrained by the availability of Coliadinae taxa with complete mitochondrial genomes; therefore, additional closely related taxa could not be included. Future research should incorporate nuclear genomic data and broader species sampling to further refine the phylogenetic framework of the genus Colias and the family Pieridae. We tested the sensitivity of the phylogenetic tree by using an alternative outgroup (Talbotia nagana); the resulting topology was completely consistent, and this alternative-outgroup tree is provided in Figure S2.

Supplementary Material

Supplemental Material
TMDN_A_2742662_SM7116.xlsx (16.5KB, xlsx)
Supplemental Material
TMDN_A_2742662_SM7115.docx (3.7MB, docx)

Acknowledgments

The authors thank Prof. Ruie Nie (Anhui Normal University, China) for assisting in the assembly of the mitogenome.

Funding Statement

This work was not supported by any funding projects.

Ethical approval

This research did not require ethical approval. Because the study subjects are invertebrate insects, their use in experiments does not require ethical clearance.

Disclosure statement

No potential conflicts of interest were reported by the authors.

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 under the accession no. PZ934385, PZ934426. All other data necessary to reported in this study can be accessed through the Dryad link: https://doi.org/10.1080/23802359.2024.2392760.

References

  1. Cao Y, Hao J, Sun X, Zheng B, Yang Q.. 2016. Molecular phylogenetic and dating analysis of pierid butterfly species using complete mitochondrial genomes. Genet Mol Res. 15(4):1–16. 10.4238/gmr15049196 [DOI] [PubMed] [Google Scholar]
  2. Carvalho APS, et al. 2024. Comprehensive phylogeny of Pieridae butterflies reveals strong correlation between diversification and temperature. IScience. 27(4):109336. 10.1016/j.isci.2024.109336 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Donath A, et al. 2019. Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Res. 47(20):10543–10552. 10.1093/nar/gkz833 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hao J, Hao J, Sun X, Zhang L, Yang Q.. 2014. The complete mitochondrial genomes of the Fenton′s wood white, Leptidea morsei, and the lemon emigrant, Catopsilia pomona. J Insect Sci. 14(1):1–22. 10.1093/jis/14.1.130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jin J, et al. 2020. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 21(1):241. 10.1186/s13059-020-02154-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Minh BQ, et al. 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 37(5):1530–1534. 10.1093/molbev/msaa015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mo S, et al. 2025. Rapid evolution of host repertoire and geographic range in a young and diverse genus of montane butterflies. Syst Biol. 74(1):141–157. 10.1093/sysbio/syae061 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Nguyen LT, Schmidt HA, Von Haeseler A, Minh BQ.. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 32(1):268–274. 10.1093/molbev/msu300 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Nie L, et al. 2018. Mitochondrial genomes of four pierid butterfly species (Lepidoptera: pieridae) with assessments about Pieridae phylogeny upon multiple mitogenomic datasets. Zool Syst. 43(4):387–409. 10.11865/zs.201834 [DOI] [Google Scholar]
  10. Ronquist F, et al. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 61(3):539–542. 10.1093/sysbio/sys029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Wahlberg N, Rota J, Braby MF, Pierce NE, Wheat CW.. 2014. Revised systematics and higher classification of pierid butterflies (Lepidoptera: pieridae) based on molecular data. Zool Scr. 43(6):641–650. 10.1111/zsc.12075 [DOI] [Google Scholar]
  12. Wang Y, et al. 2024. Complete mitochondrial genome of Colias sifanica Grum-Grshimailo, 1891 (Lepidoptera: pieridae) from montane region in northwestern China. Mitochondrial DNA B Resour. 9(8):1107–1111. 10.1080/23802359.2024.2392760 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Wang Y, Peng C, Yao Q, Shi Q, Hao J.. 2015. The complete mitochondrial genome of Gonepteryx rhamni (Lepidoptera: pieridae: coliadinae). Mitochondrial DNA. 26(5):791–792. 10.3109/19401736.2013.873930 [DOI] [PubMed] [Google Scholar]
  14. Wei F, et al. 2022. Spatio-temporal evolutionary patterns of the Pieridae butterflies (Lepidoptera: papilionoidea) inferred from mitogenomic data. Genes (Basel). 14(1):72. 10.3390/genes14010072 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Wheat CW, Watt WB, Pollock DD, Schulte PM.. 2006. From DNA to fitness differences: sequences and structures of adaptive variants of Colias phosphoglucose isomerase (PGI). Mol Biol Evol. 23(3):499–512. 10.1093/molbev/msj062 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wu CS, Xu YF (Eds.). 2017. China butterfly illustrated. Vol 1. The Straits Publishing House. [Google Scholar]
  17. Wu Y, et al. 2016. The complete mitochondrial genome of Colias erate (Lepidoptera: pieridae). Mitochondrial DNA A DNA Mapp Seq Anal. 27(6):4209–4210. 10.3109/19401736.2015.1022743 [DOI] [PubMed] [Google Scholar]
  18. Xian W, Liu Y, Ma Y, Zhou H, Song X.. 2021. Phylogenetic relationship and characterization of the complete mitochondrial genome of Colias fieldii (Lepidoptera: papilionoidea: pieridae). Mitochondrial DNA B Resour. 6(8):2146–2148. 10.1080/23802359.2021.1944379 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Xiang CY, et al. 2023. Using PhyloSuite for molecular phylogeny and tree-based analyses. Imeta. 2(1):e87. 10.1002/imt2.87 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yang J, et al. 2016. The complete mitochondrial genome of Gonepteryx mahaguru (Lepidoptera: pieridae). Mitochondrial DNA A DNA Mapp Seq Anal. 27(2):877–878. 10.3109/19401736.2014.971276 [DOI] [PubMed] [Google Scholar]
  21. Yong H, Song S, Lim P, Eamsobhana P, Suana IW.. 2017. Complete mitochondrial genome of Eurema blanda and phylogenetic relationship with its congener E. hecabe and other pierid butterflies. Meta Gene. 11:141–146. 10.3109/19401736.2015.1041116 [DOI] [Google Scholar]

Associated Data

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

Supplementary Materials

Supplemental Material
TMDN_A_2742662_SM7116.xlsx (16.5KB, xlsx)
Supplemental Material
TMDN_A_2742662_SM7115.docx (3.7MB, docx)

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 under the accession no. PZ934385, PZ934426. All other data necessary to reported in this study can be accessed through the Dryad link: https://doi.org/10.1080/23802359.2024.2392760.

Articles from Mitochondrial DNA. Part B, Resources are provided here courtesy of Taylor & Francis

RESOURCES