Next Article in Journal
Invasiveness of Impatiens parviflora in Carpathian Beech Forests: Insights from Soil Nematode Communities
Previous Article in Journal
Seasonal Variability in Non-Structural Carbohydrate Content of Warm-Adapted Zostera noltei and Zostera marina Populations
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Diversity
Volume 16
Issue 7
10.3390/d16070392
diversity-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Complete Mitochondrial Genomes of the Leptocircini Species Iphiclides podalirius and I. podalirinus (Lepidoptera: Papilionidae)

by
Yue Pan
1,2,3,†,
Xin Zhang
4,†,
Adam M. Cotton
5 and
Shao-Ji Hu
1,2,3,*
1
Institute of International Rivers and Eco-Security, Yunnan University, Kunming 650500, China
2
Yunnan Key Laboratory of International Rivers and Transboundary Eco-Security, Yunnan University, Kunming 650500, China
3
Asian International River Centre, Yunnan University, Kunming 650500, China
4
Kunming Youning Biotech Co., Ltd., Kunming 650031, China
5
86/2 Moo 5, Tambon Nong Kwai, Hang Dong, Chiang Mai, Thailand
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Diversity 2024, 16(7), 392; https://doi.org/10.3390/d16070392
Submission received: 27 May 2024 / Revised: 6 July 2024 / Accepted: 7 July 2024 / Published: 9 July 2024
(This article belongs to the Section Animal Diversity)
Browse Figures
Versions Notes

Abstract

:
The complete mitochondrial genomes of two Iphiclides species, namely I. podalirius and I. podalirinus, were sequenced, assembled, and reported in this article. Both genomes comprise 37 genes, with 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA (rRNA) genes. The gene orders and alignments agree with the reported mitogenomes of Leptocircini butterflies, while the start codon for the COX1 gene in I. podalirinus is CGA instead of the commonly seen ATN type. Codon preference shows that methionine and tryptophan are the poorest, while arginine, leucine, and serine are the richest. Phylogenetic analysis using Bayesian Inference shows both Iphiclides species are sister to the genus Lamproptera and are basal to all remaining Leptocircini species. The Kimura 2-parameter (K2P) distances of I. podalirinus from I. podalirius exceed 5%, demonstrating its solid species status. The K2P distance between the North African feisthamelii and podalirius exceeds 2%, indicating the reasonable elevation of I. feisthamelii to the full specific level as its type locality is Algeria. Future research is required to tackle the relationship between the Iberian feisthamelii and podalirius using more evidence.

1. Introduction

The genus Iphiclides Hübner [1819] is strictly Palearctic, with two generally recognised species, I. podalirius (Linnaeus, 1758), found across the majority of Eurasia, and I. podalirinus (Oberthür, 1890), restricted on the eastern margin of the Qinghai–Tibetan Plateau of West China (Figure 1) [1]. Iphiclides is one of the genera of the tribe Leptocircini, alongside the genus Lamproptera Grey, 1832 [2]. All members of the genus Lamproptera are Oriental [3], with only L. paracurius (Hu, Zhang, and Cotton, 2014) found near the boundary between the Palearctic and Oriental regions [4].
The western Mediterranean race of I. podalirius, namely feisthamelii (Duponchel, 1832), is of disputed rank (Figure 1). Regardless of its constant different wing morphology, the genetic profile of feisthamelii does not always agree with classifications. Wiemers and Gottsberger [5] reported the discordant mitochondrial and nuclear DNA pattern between podalirius and feisthamelii around the Mediterranean area, finding that only the African feisthamelii (Morocco) could be differentiated from podalirius when using mitochondrial DNA marker (COX1), while the Iberian (Spain) feisthamelii could also be differentiated when using nuclear DNA marker (EF-1α). Later, Coutsis and Oorschot [6] reported the differences in male and female genitalia between the two races and supported the elevation of feisthamelii. Based on these, Nakae [7] and Racheli and Bozano [8] listed feisthamelii as a distinct species in their recent publications.
Diversity 16 00392 g001
Figure 1. Distribution ranges of Iphiclides podalirius (red dotted line) and I. podalirinus (green dotted line), compiled from Racheli and Cotton [1], Wiemers and Gottsberger [5], and Racheli and Bozano [8], with red patches indicating the range of I. feisthamelii.
Figure 1. Distribution ranges of Iphiclides podalirius (red dotted line) and I. podalirinus (green dotted line), compiled from Racheli and Cotton [1], Wiemers and Gottsberger [5], and Racheli and Bozano [8], with red patches indicating the range of I. feisthamelii.
Diversity 16 00392 g001
Iphiclides podalirinus is distinguished from its sibling species by wing colouration; apart from the much darker ground colour and broader black bands, the red instead of yellow-orange markings on the hindwing are prominent (Figure 2). Furthermore, the distribution range of I. podalirinus is completely detached from that of I. podalirius, making it an unmistakable species (Figure 1).
In recent years, many efforts have been made to better understand the genetic diversity of I. podalirius and I. feisthamelii across Eurasia, including the whole genome data of I. podalirius published by Mackintosh, et al. [9]. However, being endemic to West China, the genetic study of I. podalirinus is still unavailable due to limited access to samples. Even though IUCN has not evaluated the threatened status of Iphiclides species yet [10], and CITES does not list them on any Appendices either [11], all species are still under much anthropogenic stress, including habitat loss, climate change, and commercialised collecting. In some European countries, I. podalirius is listed as a protected species [12]. In China, I. podalirinus is also protected by the regulation “List of Terrestrial Wildlife under State Protection for Ecological, Economic and Scientific Values” [13]. Enhancing genetic research on such species is, thus, crucial and would eventually benefit conservation [14].
The present study reports the complete mitochondrial genome of I. podalirinus, with a new genome added to I. podalirius. Our goal is to enrich the fundamental genetic data of this distinct and stylish swallowtail group in the hope of bringing new insights to its future conservation.

2. Materials and Methods

2.1. Taxon Sampling

One individual of each species, Iphiclides podalirius and I. podalirinus, was obtained by the corresponding author. Iphiclides podalirius (female) was collected from Prague, Czech Republic, in 2023, and I. podalirinus (male) was collected from western Sichuan, China, in 2019 (Figure 2). Specimens were killed immediately upon capture by pinching the thorax.
Three legs from the same side of a specimen were then removed, preserved in 95% ethanol, and stored at −20 °C until use. Specimens were spread for morphological comparison and stored in the Lepidoptera collection of the Institute of International Rivers and Eco-security, Yunnan University. Species identification of the specimens was performed primarily by morphological comparison against Racheli and Cotton [1], as the characters are very distinct for both species.
For phylogenetic reconstruction, 54 representative species of Papilionidae with complete mitochondrial genomes were selected and downloaded from the NCBI GenBank (Table 1). The 54 species cover all taxonomic groups (subfamilies and tribes) of Papilionidae. Two Pieridae species, Aporia hippia (Bremer, 1861) (OP526834) and A. bieti (Oberthür, 1884) (KX495165), were also obtained from GenBank as outgroups.
To compare the genetic differences between all Iphiclides taxa, 184 full-length (658 bp) DNA barcodes (COX1) of I. podalirius and I. feisthamelii were downloaded from the Barcode of Life Database (BOLD System v4; https://www.boldsystems.org/) (accessed on 1 April 2024) (Table 2) and analysed with the data obtained in the present study.

2.2. Molecular Work

Genomic DNA was extracted from all three legs using the Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China), then the concentration was determined on a Qubit® Flurometer (Thermo Fisher Scientific, Waltham, MA, USA), and it was stored in the refrigerator at −20 °C. The quality of the resultant DNA was examined by 1% agarose gel electrophoresis under a Gel Image Analysis System (FR-980A, Furi Science & Technology Co., Ltd., Shanghai, China).
The extracted DNA was then sequenced on an Illumina NovaSeq 6000 System (Illumina Inc., San Diego, CA, USA). The chromatograms were analysed and translated with base calling to generate raw reads in the FASTQ file. The quality of raw reads was evaluated and processed using fastp 0.36 [17] to obtain valid, high-quality, clean data.
Due to the high redundancy in high-throughput sequencing data, the de novo assembly strategy was used to align the reads and target sequences bidirectionally using SPAde 3.15 [18]. Sequencing errors were corrected first on the input sequences, and then the sequence was spliced using multiple Kmer values. Once the splicing was completed, BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 23 September 2023) was applied to compare scaffolds with the NCBI nucleotide library to assess similarity and extract the sequencing depth and coverage information of each scaffold. After filling gaps between all contigs using GapFiller 1.11 [19], the PrInSeS-G method [20] was applied to correct clipping errors and indels of small fragments during splicing. Further, built-in scripts were employed to process and evaluate contigs to obtain a complete circular genome.

2.3. Genomic Analysis

The mitochondrial genomes of Leptocircini are circular and approximately 14–16 kb in length, containing 37 genes (13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes) [21,22,23,24,25,26]. tBLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 23 September 2023) and GeneWise (https://www.ebi.ac.uk/jdispatcher/psa/genewise) (accessed on 23 September 2023) were used to perform reverse comparison with reference genomes of Graphium parus (MT198821) [24], G. eurous asakurae (MW549198) [25], and G. confucius (OK136253) [26] to obtain PCG boundaries. The tRNA regions were annotated using MiTFi [27], and CMsearch [28] was employed with the Rfam database [29] for non-coding rRNA identification. Based on the annotation and the aforementioned results, the schematics of mitochondrial genomes were mapped using Circos [30].
Codon preference plays an important role in evolution and, thus, was analysed in the present study. Relative synonymous codon usage (RSCU) was employed to measure the use frequency of codons, which shows the ratio of actual utilisation value to theoretical utilisation value. When RSCU < 1, the utilising frequency of the focal codon is lower than that of other synonymous ones; when RSCU > 1, the explanation is vice versa; and when RSCU = 1, the focal codon shows no preference [31]. The resultant RSCU plots were generated using the “ggplot2” package [32] in R 4.3.3 (http://www.r-project.org (accessed on 10 February 2024)).

2.4. Phylogeny, Genetic Distances, and BOLD BINs

To test the phylogenetic position of the obtained Iphiclides genomes against other taxonomic and systematic theories, a phylogenetic tree was reconstructed using PhyloSuite 1.2.3 [33,34] using the Iphiclides genomes with the downloaded genomes (Table 1).
ModelFinder [35] was used to estimate the best substitution model for the dataset, and Bayesian Inference (BI) was performed by MrBayes 3.2 [36] with the selected model with 10 million generation Markov Chain Monte Carlo (MCMC) (sampled every 1000th generation with a 25% burn-in) to calculate the clade posterior probabilities (PPs). The average standard deviation of split frequencies (ASDFs), the potential scale reduction factor (PSRF), and the effective sample size (ESS) were adopted to evaluate the robustness of the BI analysis. Good robustness was considered when ASDF approaches 0, PSRF approaches 1, and ESS > 200 [36,37]. The resultant tree file was annotated using FigTree 1.4.4 [38] and Adobe Illustrator CS6 (Adobe Systems Inc., San Jose, CA, USA; licensed serial number 9229-8586-7036-7176).
To estimate the degree of genetic differentiation of all Iphiclides taxa, the DNA barcode fragments (COX1) of the Iphiclides genomes were extracted and compiled with those listed in Table 2. The dataset was split into four groups in MEGA X [39] under the current taxonomic system plus geographic ranges (Figure 1), that is, I. podalirius, I. feisthamelii North Africa, I. feisthamelii Iberia, and I. podalirinus [1,7]. The Kimura 2-parameter (K2P) genetic distance [40] between groups was calculated and shown in percentage. The Barcode Index Numbers (BINs) of both I. podalirius and I. feisthamelii were also detected automatically using the web-based tool in the BOLD System (https://www.boldsystems.org/index.php/Public_BarcodeIndexNumber_Home; accessed on 2 July 2024) with the keyword “Iphiclides”.

3. Results

3.1. Sequencing Quality and Genomic Components

A total of 22.748 million reads covering 3.404 billion bases with a 150 bp average read length were obtained from the sample of I. podalirius from two reads; the ratio of Q20 reads was 98.48% and 96.50%, and that of Q30 reads was 95.28% and 90.32% in two reads, respectively. A total of 20.803 million reads covering 2.969 billion bases with a 143 bp average read length were obtained from the sample of I. podalirinus from two reads; the ratio of Q20 reads was 98.66% and 97.09%, and that of Q30 reads was 95.89% and 93.71% in two reads, respectively.
The complete mitochondrial genome of I. podalirius is 15,166 bp in length (PP566972), and that of I. podalirinus is 15,229 bp in length (PP566973). Both genomes are circular and contain 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, and 2 ribosomal RNA genes (Figure 3). Among the 13 PCGs, ND2, COX1, COX2, ATP8, ATP6, COX3, ND3, ND6, and CYTB are coded by plus strands, while ND5, ND4, ND4L, and ND1 are coded by minus strands (Table 3). The CG content of the mitogenome of I. podalirius is 18.1%, and that of the mitogenome of I. podalirinus is 19.1%.

3.2. Codon Usage, Preference, and Substitution

Both species share the same set of anti-codons for all 22 transfer RNAs. For I. podalirius, 12 out of 13 start codons are the ATN type, while only one of the ND5 gene is a specialised TTG; 8 out of 13 stop codons are the typical TAA; and two (ND3 and ND1) are TAG. The stop codons of COX1, COX2, and ND4 are incomplete and inferred to be compensated by the 3′-adenine residuals to the mRNA. For I. podalirinus, the usage of both start and stop codons is very similar to those of I. podalirius in general, while the start codon of COX1 is CGA and the stop codon of ND3 is TAA instead of TAG (Table 3).
The codon preference analysis showed that both methionine and tryptophan possess only one codon each, being the poorest, while arginine, leucine, and serine possess five codons each, being the richest. Among the remaining amino acids, nine are coded by two codons, one is coded by three codes, and five are coded by four codes. The codon usage and preferences of I. podalirius and I. podalirinus are shown in Figure 4.

3.3. Phylogenetic Validation, Genetic Distance, and BINs

The BI analysis resulted in an ASDF = 0.000207, an average PSRF = 1.000 (maximal 1.001), and an average ESS = 6645.74 for all parameters, indicating the runs have reached excellent robustness.
The phylogenetic tree reconstructed by Bayesian Inference produced a robust topology and clear division of all subfamilies and tribes in Papilionidae, as reported by Condamine, et al. [2], with most node values (PPs) in the backbone of subfamilies and tribes exceeding 0.90, if not maximal. Within each tribal clade, most nodes were supported by maximal PP values, with only that between genera Iphiclides and Lamproptera being the lowest (0.85) (Figure 5 and Figure S1 in Supplementary Materials).
The two Iphiclides species are sister to each other and were altogether placed sister to genus Lamproptera at the basal part of tribe Leptocircini (Figure 5), reflecting the taxonomic position of genus Iphiclides based on morphological characters.
The K2P distances of DNA barcode (COX1) between I. podalirinus and the remaining taxa reached 5.280–5.530%, being the largest, and that between I. podalirius and the Iberian I. feisthamelii was only 0.134%, being the smallest (Table 4). The K2P distances between the North African I. feisthamelii and both I. podalirius and the Iberian I. feisthamelii are intermediate, being 2.184–2.234% (Table 4). The BIN search for “Iphiclides” from BOLD data collection yielded two BINs, with North African I. feisthamelii being one distinct BIN (25 sequences), while the typical I. podarilius and Iberian I. feisthamelii together being another BIN (198 sequences) (accessed on 2 July 2024) (Table 5).

4. Discussion

The two Iphiclides species share the same mitochondrial genomic configurations with each other, as do most other Leptocircini butterflies [21,23,24,25,26]. The most frequently used codons are TTA and AGA, and codons in the third position in A or T reflect the AT bias of the insect mitochondrial genome. It is noticeable that in the mitogenome of I. podalirinus, the start codon for the COX1 gene is CGA instead of the regular ATN type (Table 3). Kim, et al. [41] argued that the CGA start codon (arginine) could be a synapomorphic character of arthropods, and Papilionidae species used in this study, such as Parnassius stubbendorfii, Sericinus montela, Graphium sarpedon, Papilio xuthus, and P. elwesi, all possess this start codon for the COX1 gene. Future studies are required to tackle the origin of the uncertainty of the start codon for the COX1 gene in Lepidoptera and integrate the start codons of more species.
Molecular studies have been instrumental in supporting and revising taxonomies based on morphology and geographical distribution. Our analysis shows that the K2P genetic distances between I. podalirinus and the remaining taxa of this genus are all over 5% (Table 3), demonstrating its solid species status, as represented by morphological differences on the wings. However, the genetic differences between I. feisthamelii and I. podalirius are interesting. For the North African feisthamelii, the genetic distance between it and I. podalirius reached 2.234% (Table 4), indicating its distinct species status when one takes 2% as the threshold for species delimitation [42]. On the contrary, the Iberian feisthamelii is much closer to I. podalirius genetically (Table 4). BOLD BIN search also agreed with this delimitation (Table 5). Similar discordance has been reported in the Iberian Melitaea species when nuclear gene markers performed better in species delimitation than using mitochondrial gene markers alone [43].
Given that the type locality of feisthamelii is Algeria in North Africa [44], it is reasonable to treat it as a distinct species with reported morphological and genitalic differences [6], as well as the genetic differentiations reported for either mitochondrial or nuclear genes. However, the status of Iberian feisthamelii has long been obscured due to the paradox in its genetic relationship with I. podalirius using different genetic markers [5] until Gaunet, et al. [45] discovered such discordance is the result of genetic sweeping and mitochondrial introgression due to repeated Wolbachia infections.
Wolbachia is a type of inheritable Rickettsia-like endosymbiont that can infect insects with certain taxonomic specialisations [46]. Populations infected by Wolbachia either exhibit a modified mitochondrial genetic profile, which could affect subsequent analysis of population genetics, demographic history, and even phylogenetic relationships [45,47]. Hence, future taxonomies involving molecular evidence must either evaluate the Wolbachia infection among samples or include sufficient nuclear genes to avoid obscure results hindering our recognition of distinctive taxa [48,49].

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d16070392/s1, Figure S1: The Bayesian Inference phylogenetic tree with numeric node values and outgroups.

Author Contributions

Conceptualization, S.-J.H. and X.Z.; methodology, X.Z.; software, X.Z. and S.-J.H.; formal analysis, Y.P., X.Z. and S.-J.H.; resources, S.-J.H., A.M.C. and X.Z.; data curation, S.-J.H.; writing—original draft preparation, Y.P. and X.Z.; writing—review and editing, S.-J.H. and A.M.C.; visualisation, Y.P., S.-J.H. and A.M.C.; supervision, S.-J.H. and A.M.C.; project administration, S.-J.H.; funding acquisition, S.-J.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Academician (Expert) Working Station of the Yunnan Province Science and Technology Department, grant number 202305AF150037, the NSFC Programme of China, grant number 41761011, and the Biodiversity Conservation Programme of the Ministry of Ecology and Environment, China (China-BON Butterflies), grant number SDZXWJZ01013.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All mitochondrial genomes used in the present study are openly available from NCBI GenBank via the accession numbers listed in Table 1. All DNA barcodes (COX1) used in the present study from the BOLD System are listed in Table 2.

Acknowledgments

The authors thank Yang Yang (Beijing, China) for his assistance in obtaining samples.

Conflicts of Interest

Xin Zhang was employed by the company Kunming Youning Biotech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Racheli, T.; Cotton, A.M. Guide to the Butterflies of the Palearctic Region. Papilionidae Part I. Subfamily Papilioninae. Tribes Leptocircini Teinopalpini; Omnes Artes: Milano, Italy, 2009; p. 69. [Google Scholar]
  2. Condamine, F.L.; Nabholz, B.; Clamens, A.-L.; Dupuis, J.R.; Sperling, F.A.H. Mitochondrial phylogenomics, the origin of swallowtail butterflies, and the impact of the number of clocks in Bayesian molecular dating. Syst. Entomol. 2018, 43, 460–480. [Google Scholar] [CrossRef]
  3. Tsukada, E.; Nishiyama, Y. Butterflies of the South East Asian Islands. Part I. Papilionidae; Plapac Co.: Tokyo, Japan, 1980; p. 457. [Google Scholar]
  4. Hu, S.J.; Cotton, A.M.; Lamas, G.; Duan, K.; Zhang, X. Checklist of Yunnan Papilionidae (Lepidoptera: Papilionoidea) with nomenclatural notes and descriptions of new subspecies. Zootaxa 2023, 5362, 1–69. [Google Scholar] [CrossRef] [PubMed]
  5. Wiemers, M.; Gottsberger, B. Discordant patterns of mitochondrial and nuclear differentiation in the Scarce Swallowtail Iphiclides podalirius feisthamelii (Duponchel, 1832) (Lepidoptera: Papilionidae). Entomol. Z. Stuttg. 2010, 120, 111–115. [Google Scholar]
  6. Coutsis, J.G.; Oorschot, H.v. Differences in the male and female genitalia between Iphiclides podalirius and Iphiclides feisthamelii, further supporting species status for the latter (Lepidoptera: Papilionidae). Phegea 2011, 39, 12–22. [Google Scholar]
  7. Nakae, M. Papilionidae of the World; Roppon-Ashi: Tokyo, Japan, 2021; p. 336. [Google Scholar]
  8. Racheli, T.; Bozano, G.C. Guide to the Butterflies of the Palearctic Region. Papilionidae Part I. Subfamily Papilioninae. Tribes Leptocircini Teinopalpini, 2nd ed.; Omnes Artes: Milano, Italy, 2024. [Google Scholar]
  9. Mackintosh, A.; Laetsch, D.R.; Baril, T.; Ebdon, S.; Jay, P.; Vila, R.; Hayward, A.; Lohse, K. The genome sequence of the scarce swallowtail, Iphiclides podalirius. G3 2022, 12, jkac193. [Google Scholar] [CrossRef]
  10. IUCN. The IUCN Red List of Threatened SpeciesTM. Available online: https://www.iucnredlist.org/search?query=Iphiclides&searchType=species (accessed on 5 April 2024).
  11. CITES. The CITES Appendices I, II, and III. Available online: https://cites.org/eng/app/appendices.php (accessed on 5 April 2024).
  12. Collins, N.M.; Morris, M.G. Threatened Swallowtail Butterflies of the World: The IUCN Red Data Book; IUCN: Gland, Switzerland; Cambridge, UK, 1985; p. 331. [Google Scholar]
  13. NFGA. List of Terrestrial Wildlife under State Protection for Ecological, Economic and Scientific Values (Bulletin of the State Forestry Administration of P. R. China 2000-07); National Forestry and Grassland Administration: Beijing, China, 2000.
  14. Wang, W.L.; Suman, D.O.; Zhang, H.H.; Xu, Z.B.; Ma, F.Z.; Hu, S.J. Butterfly conservation in China: From science to action. Insects 2020, 11, 661. [Google Scholar] [CrossRef] [PubMed]
  15. Shi, M.R.; Yu, H.; Xu, J. The complete mitochondrial genome of the Papilio memno [sic!] (Lepidoptera: Papilionidae). Mitochondrial DNA Part B 2020, 5, 2159–2160. [Google Scholar] [CrossRef]
  16. Condamine, F.L.; Allio, R.; Reboud, E.L.; Dupuis, J.R.; Toussaint, E.F.A.; Mazet, N.; Hu, S.J.; Lewis, D.S.; Kunte, K.; Cotton, A.M.; et al. A comprehensive phylogeny and revised taxonomy illuminate the origin and diversification of the global radiation of Papilio (Lepidoptera: Papilionidae). Mol. Phylogenetic Evol. 2023, 183, 107758. [Google Scholar] [CrossRef]
  17. Chen, S.F.; Zhou, Y.Q.; Chen, Y.R.; Gu, J. fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018, 34, i884–i890. [Google Scholar] [CrossRef]
  18. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.; Nikolenko, S.; Pham, S.; Prjibelski, A.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef]
  19. Boetzer, M.; Pirovano, W. Toward almost closed genomes with GapFiller. Genome Biol. 2012, 13, R56. [Google Scholar] [CrossRef] [PubMed]
  20. Massouras, A.; Hens, K.; Gubelmann, C.; Uplekar, S.; Decouttere, F.; Rougemont, J.; Cole, S.T.; Deplancke, B. Primer-initiated sequence synthesis to detect and assemble structural variants. Nat. Methods 2010, 7, 485–486. [Google Scholar] [CrossRef] [PubMed]
  21. Qin, X.M.; Guan, Q.X.; Li, H.M.; Zhang, Y.; Liu, Y.J.; Guo, D.N. The complete mitogenome of Lamproptera curia (Lepidoptera: Papilionidae) and phylogenetic analyses of Lepidoptera. Front. Biol. 2015, 10, 458–472. [Google Scholar] [CrossRef]
  22. Chen, X.; Hao, J.S. The complete mitochondrial genome of Graphium chironides (Lepidoptera: Papilionidae: Papilioninae). Mitochondrial DNA 2015, 27, 1–3. [Google Scholar] [CrossRef] [PubMed]
  23. Kong, W.J.; Hou, Y.; Zhang, G.T.; Yang, T.; Xiao, R.H. Complete mitochondrial genome of Graphium doson (Papilioninae: Leptocircini). Mitochondrial DNA Part B 2019, 4, 698–699. [Google Scholar] [CrossRef]
  24. Duan, K.; Zhang, X.; Zhang, H.H.; Hu, S.J. Complete mitochondrial genome of the subalpine swordtail butterfly Graphium (Pazala) parus (Nicéville, 1900) (Lepidoptera: Papilionidae). Mitochondrial DNA Part B 2020, 5, 1903–1904. [Google Scholar] [CrossRef]
  25. Hu, S.J.; Zhang, X.; Duan, K. Complete mitochondrial genomes of two insular races of Pazala swordtails from Taiwan, China (Lepidoptera: Papilionidae: Graphium). Mitochondrial DNA Part B 2021, 6, 1557–1559. [Google Scholar] [CrossRef] [PubMed]
  26. He, F.R.; Zhang, X.; Hu, S.J. Complete mitochondrial genome of the recently discovered multivoltine Graphium (Pazala) confucius Hu, Duan & Cotton, 2018 (Lepidoptera: Papilionidae). Mitochondrial DNA Part B 2022, 7, 138–140. [Google Scholar] [CrossRef] [PubMed]
  27. Jühling, F.; Pütz, J.; Bernt, M.; Donath, A.; Middendorf, M.; Florentz, C.; Stadler, P.F. Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements. Nucleic Acids Res. 2012, 40, 2833–2845. [Google Scholar] [CrossRef]
  28. Cui, X.F.; Liu, Z.W.; Wang, S.; Wang, J.J.-Y.; Gao, X. CMsearch: Simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction. Bioinformatics 2016, 32, i332–i340. [Google Scholar] [CrossRef]
  29. Kalvari, I.; Nawrocki, E.P.; Argasinska, J.; Quinones-Olvera, N.; Finn, R.D.; Bateman, A.; Petrov, A.I. Non-coding RNA analysis using the Rfam database. Curr. Protoc. Bioinform. 2018, 62, e51. [Google Scholar] [CrossRef]
  30. Krzywinski, M.; Schein, J.; Birol, İ.; Connors, J.; Gascoyne, R.; Horsman, D.; Jones, S.J.; Marra, M.A. Circos: An information aesthetic for comparative genomics. Genome Res. 2009, 19, 1639–1645. [Google Scholar] [CrossRef] [PubMed]
  31. Yu, X.L.; Liu, J.X.; Li, H.Z.; Liu, B.Y.; Zhao, B.Q.; Ning, Z.Y. Comprehensive analysis of synonymous codon usage patterns and influencing factors of porcine epidemic diarrhea virus. Arch. Virol. 2021, 166, 157–165. [Google Scholar] [CrossRef]
  32. Wickham, H. ggplot2: Elegant Graphics for Data Analysis; Springer: New York, NY, USA, 2016. [Google Scholar]
  33. Zhang, D.; Gao, F.L.; Jakovlić, I.; Zou, H.; Zhang, J.; Li, W.X.; Wang, G.T. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol. Ecol. Resour. 2020, 20, 348–355. [Google Scholar] [CrossRef] [PubMed]
  34. Xiang, C.Y.; Gao, F.L.; Jakovlić, I.; Lei, H.P.; Hu, Y.; Zhang, H.; Zou, H.; Wang, G.T.; Zhang, D. Using PhyloSuite for molecular phylogeny and tree-based analyses. iMeta 2023, 2, e87. [Google Scholar] [CrossRef] [PubMed]
  35. Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.F.; von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 2017, 14, 587–589. [Google Scholar] [CrossRef]
  36. Ronquist, F.; Teslenko, M.; Van Der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M.A.; Huelsenbeck, J.P. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012, 61, 539–542. [Google Scholar] [CrossRef] [PubMed]
  37. Gelman, A.; Rubin, D.B. Inference from iterative simulation using multiple sequences. Stat. Sci. 1992, 7, 457–472. [Google Scholar] [CrossRef]
  38. Rambaut, A. Figtree ver 1.4.4. Institute of Evolutionary Biology; University of Edinburgh: Edinburgh, UK, 2018. [Google Scholar]
  39. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547. [Google Scholar] [CrossRef]
  40. Kimura, M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980, 16, 111–120. [Google Scholar] [CrossRef]
  41. Kim, M.I.; Baek, J.Y.; Kim, M.J.; Jeong, H.C.; Kim, K.G.; Bae, C.H.; Han, Y.S.; Jin, B.R.; Kim, I. Complete nucleotide sequence and organization of the mitogenome of the red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) and comparison with other lepidopteran insects. Mol. Cells 2009, 28, 347–363. [Google Scholar] [CrossRef] [PubMed]
  42. Meyer, C.P.; Paulay, G. DNA barcoding: Error rates based on comprehensive sampling. PLoS Biol. 2005, 3, e422. [Google Scholar] [CrossRef] [PubMed]
  43. Hinojosa, J.C.; Tóth, J.P.; Monasterio, Y.; Mesa, L.S.; Sariot, M.G.M.; Escobés, R.; Vila, R. Integrative taxonomy reveals a new Melitaea (Lepidoptera: Nymphalidae) species widely distributed in the Iberian Peninsula. Insect Syst. Divers. 2022, 6, ixac004. [Google Scholar] [CrossRef]
  44. Duponchel, P.A.J. Supplement. Diurnes. In Histoire naturelle des Lépidoptèrs ou Papillons de France; Godart, J.B., Ed.; Méquignon-Marvis, Libraire-Éditeur: Paris, France, 1832; pp. 1–466. [Google Scholar]
  45. Gaunet, A.; Dincă, V.; Dapporto, L.; Montagud, S.; Vodă, R.; Schär, S.; Badiane, A.; Font, E.; Vila, R. Two consecutive Wolbachia-mediated mitochondrial introgressions obscure taxonomy in Palearctic swallowtail butterflies (Lepidoptera, Papilionidae). Zool. Scr. 2019, 48, 507–519. [Google Scholar] [CrossRef]
  46. Werren, J.H.; Baldo, L.; Clark, M.E. Wolbachia: Master manipulators of invertebrate biology. Nat. Rev. Microbiol. 2008, 6, 741–751. [Google Scholar] [CrossRef]
  47. Hurst, G.D.D.; Jiggins, F.M. Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: The effects of inherited symbionts. Proc. R. Soc. B Biol. Sci. 2005, 272, 1525–1534. [Google Scholar] [CrossRef]
  48. Baldo, L.; Werren, J.H. Revisiting Wolbachia supergroup typing based on WSP: Spurious lineages and discordance with MLST. Curr. Microbiol. 2007, 55, 81–87. [Google Scholar] [CrossRef] [PubMed]
  49. Lü, J.; Hu, S.J.; Ma, X.Y.; Chen, J.M.; Li, Q.Q.; Ye, H. Origin and expansion of the Yunnan Shoot Borer, Tomicus yunnanensis (Coleoptera: Scolytinae): A mixture of historical natural expansion and contemporary human-mediated relocation. PLoS ONE 2014, 9, e111940. [Google Scholar] [CrossRef]
Diversity 16 00392 g002
Figure 2. The female Iphiclides podalirius (A) and male I. podalirinus (B) specimens used in this study, upperside on the left side and half of the underside on the right side; scale bar = 10 mm.
Figure 2. The female Iphiclides podalirius (A) and male I. podalirinus (B) specimens used in this study, upperside on the left side and half of the underside on the right side; scale bar = 10 mm.
Diversity 16 00392 g002
Diversity 16 00392 g003
Figure 3. Mitochondrial genomic schematics of Iphiclides podalirius (A) and I. podalirinus (B) assembled in this study.
Figure 3. Mitochondrial genomic schematics of Iphiclides podalirius (A) and I. podalirinus (B) assembled in this study.
Diversity 16 00392 g003
Diversity 16 00392 g004
Figure 4. RSCU scores of codon usage in the mitochondrial genomes of Iphiclides podalirius (A) and I. podalirinus (B).
Figure 4. RSCU scores of codon usage in the mitochondrial genomes of Iphiclides podalirius (A) and I. podalirinus (B).
Diversity 16 00392 g004
Diversity 16 00392 g005
Figure 5. The phylogenetic tree of representatives of all subfamilies and tribes in Papilionidae using Bayesian Inference (BI); outgroups are not shown; circles at the nodes represent node values (PPs); and the tip names of the focal Iphiclides species are in bold font. Pictures of representative species for each tribe were photographed and edited by Shao-Ji Hu and Adam M. Cotton. Pictures are not proportional to actual butterfly sizes.
Figure 5. The phylogenetic tree of representatives of all subfamilies and tribes in Papilionidae using Bayesian Inference (BI); outgroups are not shown; circles at the nodes represent node values (PPs); and the tip names of the focal Iphiclides species are in bold font. Pictures of representative species for each tribe were photographed and edited by Shao-Ji Hu and Adam M. Cotton. Pictures are not proportional to actual butterfly sizes.
Diversity 16 00392 g005
Table 1. Mitochondrial genomes of Papilionidae representative species used in the present study.
Table 1. Mitochondrial genomes of Papilionidae representative species used in the present study.
SubfamilyTribeSpeciesGenBank Accession
BaroniinaeBaroniiniBaronia brevicornisOR063968
ParnassiinaeSericininiAllancastria cerisyLS974636
Bhutanitis mansfieldiOP936023
Sericinus montelaMN013020
LuehdorfiiniLuehdorfia chinensisOR058734
ParnassiiniParnassius epaphusKM373898
Parnassius stubbendorfiiOP709281
Parnassius hardwickiiOP963809
Parnassius stoliczkanusOP963808
Parnassius imperatorKM507326
PapilioninaeLeptocirciniLamproptera megesLT999978
Lamproptera curiusKJ141168
Graphium parusMT198821
Graphium mullahKJ472924
Graphium eurous asakuraeMW549198
Graphium confuciusOK136253
Graphium antiphatesON620258
Graphium agetesOQ935417
Graphium leechiKX011066
Graphium chironidesKP159289
Graphium dosonMK144328
Graphium agamemnonOQ852718
Graphium sarpedonON675575
Graphium cloanthusOQ852719
TeinopalpiniTeinopalpus imperialisKR018842
Teinopalpus aureusHM563681
TroidiniCressida cressidaMK507889
Euryades corethrusLT999972
Losaria neptunusLT999979
Pachliopta kotzebueaLS975120
Pachliopta aristolochiaeKU950357
Atrophaneura dixoniLT999977
Byasa polyeuctesOQ673106
Byasa alcinousKJ540880
Trogonoptera brookianaLT999986
Troides aeacusEU625344
Ornithoptera priamusLT999981
Ornithoptera alexandraeOQ590009
PapilioniniPapilio machaonHM243594
Papilio xuthusKT922004
Papilio rexKX033354
Papilio dardanusKX033351
Papilio demoleusKR024009
Papilio parisOQ927990
Papilio bianorJN019809
Papilio syfaniusKJ396621
Papilio maackiiKC433408
Papilio polytesKM215138
Papilio helenusKP247522
Papilio protenorKY272622
Papilio macilentusOP324646
Papilio agenor 1MH981597
Papilio janaka 2OQ955740
Papilio alcmenorON964516
1 Papilio memnon in the GenBank record. 2 Papilio bootes janaka in the GenBank record. Based on the specimen information in GenBank records and the latest taxonomic research on the genus Papilio [15,16], the species names Papilio agenor and Papilio janaka were adopted herein.
Table 2. The 184 DNA barcode (COX1) sequences of I. podalirius and I. feisthamelii downloaded from BOLD for this study.
Table 2. The 184 DNA barcode (COX1) sequences of I. podalirius and I. feisthamelii downloaded from BOLD for this study.
TaxonCountryBOLD Accession
I. podaliriusPortugalBDE369-19, EULEP5983-18, EULEP5984-18
SpainEULEP5951-18, EULEP5952-18, EULEP5980-18, EULEP5994-18, WMB3432-14
FranceBIBSA922-15, BIBSA2258-20, BIBSA2271-20, EULEP4099-16, EULEP5979-18, EULEP5986-18, EULEP5987-18, EULEP5988-18, EULEP5989-18, EULEP5992-18, EZSPM448-09, OXB1065-15, OXB1259-15, OXB1366-15, OXB1388-15, WMB354-11, WMB1173-13, WMB1730-13, WMB1800-13, WMB3465-14, WMB3893-14, WMB3912-14, WMB3916-14, WMB3945-14, WMB3971-14, WMB4637-14, WMB5327-14
SwitzerlandEULEP2164-15, EULEP4098-16
GermanyGWORA2430-09
AustriaABOLC133-16, ABOLD084-16, LEASS703-17, LEASS856-17
ItalyBIBSA349-15, BIBSA580-15, BIBSA701-15, BIBSA970-15, BIBSA1031-15, BIBSA1140-15, BIBSA1171-15, BIBSA1329-15, BIBSA1423-15, BIBSA1600-16, BIBSA1613-16, BIBSA1618-16, BIBSA1667-16, BIBSA1766-16, EULEP5662-17, EULEP5953-18, EULEP5961-18, EULEP5965-18, EULEP5982-18, EULEP5991-18, EULEP5999-18, EULEP6000-18, LEATD016-13, LEATG462-14, LEASZ969-22, OXB587-15, OXB888-15, OXB910-15, OXB974-15, OXB1028-15, WMB127-11, WMB259-11, WMB641-11, WMB1895-13, WMB2120-13, WMB2216-13, WMB2360-13, WMB2361-13, WMB2442-13, WMB3834-14, WMB3853-14, WMB4131-14, WMB4271-14, WMB4288-14, WMB4342-14, WMB4765-14, WMB4804-14, WMB4855-14, WMB4968-14, WMB5040-14, WMB5084-14WMB5497-14, WMB6563-18
SlovakiaEULEP5022-16
UkraineEULEP4835-16
RomaniaEULEP001-14, EULEP5973-18, EZRMN332-08, EZROM219-08, EZROM220-08, EZROM351-08, EZROM1028-08, EZROM1029-08, EZROM1030-08
BulgariaEULEP897-15, EULEP1197-15
GreeceEULEP701-15, EULEP1306-15, EULEP1367-15, EULEP1442-15, EULEP1600-15, EULEP1767-15, EULEP5962-18, EULEP5971-18, EULEP5972-18, EULEP5974-18, EULEP5975-18, EULEP5978-18
TurkeyEULEP5950-18
IranIRANB315-08
KazakhstanEULEP5956-18, EULEP5957-18, EULEP5958-18, EULEP5959-18, EULEP5960-18
RussiaLEFIL147-10
I. feisthameliiMoroccoEULEP5954-18, EULEP5955-18, EULEP5990-18, EULEP5996-18, EULEP5997-18, WMB245-11, WMB373-11, WMB399-11, WMB421-11, WMB3604-14, BDE293-19, BDE293-19, BDE294-19
AlgeriaEULEP5964-18, EULEP5981-18, EULEP6001-18, WMB1472-13, WMB1473-13, WMB1476-13
TunisiaEULEP5970-18, WMB1405-13, WMB1406-13, WMB6562-18
PortugalWMB4400-14, BDE553-19
SpainEULEP5949-18, EULEP5966-18, EULEP5967-18, EULEP5968-18, EULEP5969-18, EULEP5993-18, EULEP5995-18, EULEP5998-18, EULEP6002-18, EZSPC449-09, EZSPC450-09, EZSPC451-09, EZSPM099-09, EZSPM275-09, EZSPM362-09, EZSPM419-09, EZSPM450-09, EZSPM611-12, EZSPM629-12, EZSPN330-09, EZSPN403-09, EZSPN477-09, EZROM725-08, WMB3126-14, WMB3315-14, WMB3481-14, WMB3777-14, WMB3995-14, WMB4046-14
FranceEULEP002-14, EULEP003-14, EULEP5985-18
Table 3. The basic components, strand properties of each gene, anti-codon of tRNA, and start and stop codons of 13 protein-coding genes of the mitochondrial genomes of Iphiclides podalirius (A) and I. podalirinus (B).
Table 3. The basic components, strand properties of each gene, anti-codon of tRNA, and start and stop codons of 13 protein-coding genes of the mitochondrial genomes of Iphiclides podalirius (A) and I. podalirinus (B).
GeneStrandSpanAnti-CodonStart CodonStop Codon
ABABAB
tRNA-Met+1–661–67CAU————
tRNA-Ile+67–13068–131GAU————
tRNA-Gln−128–196129–197UUG————
ND2+243–1256256–1296—ATTATTTAATAA
tRNA-Trp+1255–13191268–1332UCA————
tRNA-Cys−1312–13791325–1391GCA————
tRNA-Tyr−1380–14441394–1464GUA————
COX1+1445–29781468–2998—ATGCGA 1T-tRNAT-tRNA
tRNA-Leu+2979–30452999–3065UAA————
COX2+3046–37273066–3747—ATGATGT-tRNAT-tRNA
tRNA-Lys+3728–37973748–3817CUU————
tRNA-Asp+3798–38653818–3884GUC————
ATP8+3866–40243885–4043—ATCATATAATAA
ATP6+4018–46954037–4714—ATGATGTAATAA
COX3+4701–54924720–5508—ATAATATAATAA
tRNA-Gly+5496–55615512–5577UCC————
ND3+5562–59155578–5931—ATTATATAGTAA
tRNA-Ala+5914–59765931–5993UGC————
tRNA-Arg+5977–60395994–6057UCG————
tRNA-Asn+6041–61056059–6123GUU————
tRNA-Ser+6107–61676125–6187UCU————
tRNA-Glu+6183–62476197–6263UUC————
tRNA-Phe−6246–63106262–6326GAA————
ND5−6311–80506327–8066—TTGTTGTAATAA
tRNA-His−8051–81168067–8132GUG————
ND4−8116–94558132–9471—ATGATGT-tRNAT-tRNA
ND4L−9456–97469472–9762—ATGATGTAATAA
tRNA-Thr+9749–98139765–9828UGU————
tRNA-Pro−9814–98789829–9893UGG————
ND6+9881–10,4119896–10,426—ATTATTTAATAA
CYTB+10,416–1156410,431–11,579—ATGATGTAATAA
tRNA-Ser+11,580–11,64711,597–11,665UGA————
ND1−11,665–12,60311,683–12,621—ATGATGTAGTAG
tRNA-Leu−12,605–12,67212,623–12,689UAG————
16S-rRNA+12,691–14,02912,709–14,056—————
tRNA-Val−14,028–14,09714,055–14,126UAC————
12S-rRNA+14,098–14,89514,127–14,900—————
1 Start codon not determined.
Table 4. The Kimura 2-parameter (K2P) genetic distances between I. podalirius, I. feisthamelii (two geographic groups), and I. podalirinus in percentage.
Table 4. The Kimura 2-parameter (K2P) genetic distances between I. podalirius, I. feisthamelii (two geographic groups), and I. podalirinus in percentage.
12a2b3
1. I. podalirius
2a. I. feisthamelii North Africa2.234
2b. I. feisthamelii Iberia0.1342.184
3. I. podalirinus5.3095.5305.280
Table 5. The Barcode Index Numbers (BINs) search result using “Iphiclides” in the BOLD System; the numbers in parentheses after country/region names indicate the number of sequences contained.
Table 5. The Barcode Index Numbers (BINs) search result using “Iphiclides” in the BOLD System; the numbers in parentheses after country/region names indicate the number of sequences contained.
BIN NumberNo. of SequencesSpecies IncludedCountry/Region
BOLD:AAB2272198I. podalirius
I. feisthamelii		Italy (58); Spain (40); France (34); Greece (16); Romania (10); Portugal (7); Austria (6); Switzerland (5); Kazakhstan (5); Czech Republic (3); Iran (3); Russia (2); Slovakia (2); Bulgaria (2); Turkey (2); Germany (1); Ukraine (1); Unknown (1)
BOLD:AEA309825I. feisthameliiMorocco (13); Algeria (7); Tunisia (4); Unknown (1)
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Pan, Y.; Zhang, X.; Cotton, A.M.; Hu, S.-J. Complete Mitochondrial Genomes of the Leptocircini Species Iphiclides podalirius and I. podalirinus (Lepidoptera: Papilionidae). Diversity 2024, 16, 392. https://doi.org/10.3390/d16070392

AMA Style

Pan Y, Zhang X, Cotton AM, Hu S-J. Complete Mitochondrial Genomes of the Leptocircini Species Iphiclides podalirius and I. podalirinus (Lepidoptera: Papilionidae). Diversity. 2024; 16(7):392. https://doi.org/10.3390/d16070392

Chicago/Turabian Style

Pan, Yue, Xin Zhang, Adam M. Cotton, and Shao-Ji Hu. 2024. "Complete Mitochondrial Genomes of the Leptocircini Species Iphiclides podalirius and I. podalirinus (Lepidoptera: Papilionidae)" Diversity 16, no. 7: 392. https://doi.org/10.3390/d16070392

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop