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

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. 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 omitted. See PDF]

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].

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.

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.

Footnotes

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Figures and Tables

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 3. Mitochondrial genomic schematics of Iphiclides podalirius (A) and I. podalirinus (B) assembled in this study.

Figure 4. RSCU scores of codon usage in the mitochondrial genomes of Iphiclides podalirius (A) and I. podalirinus (B).

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.

Table 1

Mitochondrial genomes of Papilionidae representative species used in the present study.

Subfamily	Tribe	Species	GenBank Accession
Baroniinae	Baroniini	Baronia brevicornis	OR063968
Parnassiinae	Sericinini	Allancastria cerisy	LS974636
		Bhutanitis mansfieldi	OP936023
		Sericinus montela	MN013020
	Luehdorfiini	Luehdorfia chinensis	OR058734
	Parnassiini	Parnassius epaphus	KM373898
		Parnassius stubbendorfii	OP709281
		Parnassius hardwickii	OP963809
		Parnassius stoliczkanus	OP963808
		Parnassius imperator	KM507326
Papilioninae	Leptocircini	Lamproptera meges	LT999978
		Lamproptera curius	KJ141168
		Graphium parus	MT198821
		Graphium mullah	KJ472924
		Graphium eurous asakurae	MW549198
		Graphium confucius	OK136253
		Graphium antiphates	ON620258
		Graphium agetes	OQ935417
		Graphium leechi	KX011066
		Graphium chironides	KP159289
		Graphium doson	MK144328
		Graphium agamemnon	OQ852718
		Graphium sarpedon	ON675575
		Graphium cloanthus	OQ852719
	Teinopalpini	Teinopalpus imperialis	KR018842
		Teinopalpus aureus	HM563681
	Troidini	Cressida cressida	MK507889
		Euryades corethrus	LT999972
		Losaria neptunus	LT999979
		Pachliopta kotzebuea	LS975120
		Pachliopta aristolochiae	KU950357
		Atrophaneura dixoni	LT999977
		Byasa polyeuctes	OQ673106
		Byasa alcinous	KJ540880
		Trogonoptera brookiana	LT999986
		Troides aeacus	EU625344
		Ornithoptera priamus	LT999981
		Ornithoptera alexandrae	OQ590009
	Papilionini	Papilio machaon	HM243594
		Papilio xuthus	KT922004
		Papilio rex	KX033354
		Papilio dardanus	KX033351
		Papilio demoleus	KR024009
		Papilio paris	OQ927990
		Papilio bianor	JN019809
		Papilio syfanius	KJ396621
		Papilio maackii	KC433408
		Papilio polytes	KM215138
		Papilio helenus	KP247522
		Papilio protenor	KY272622
		Papilio macilentus	OP324646
		Papilio agenor 1	MH981597
		Papilio janaka 2	OQ955740
		Papilio alcmenor	ON964516

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.

Taxon	Country	BOLD Accession
I. podalirius	Portugal	BDE369-19, EULEP5983-18, EULEP5984-18
	Spain	EULEP5951-18, EULEP5952-18, EULEP5980-18, EULEP5994-18, WMB3432-14
	France	BIBSA922-15, 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
	Switzerland	EULEP2164-15, EULEP4098-16
	Germany	GWORA2430-09
	Austria	ABOLC133-16, ABOLD084-16, LEASS703-17, LEASS856-17
	Italy	BIBSA349-15, 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
	Slovakia	EULEP5022-16
	Ukraine	EULEP4835-16
	Romania	EULEP001-14, EULEP5973-18, EZRMN332-08, EZROM219-08, EZROM220-08, EZROM351-08, EZROM1028-08, EZROM1029-08, EZROM1030-08
	Bulgaria	EULEP897-15, EULEP1197-15
	Greece	EULEP701-15, EULEP1306-15, EULEP1367-15, EULEP1442-15, EULEP1600-15, EULEP1767-15, EULEP5962-18, EULEP5971-18, EULEP5972-18, EULEP5974-18, EULEP5975-18, EULEP5978-18
	Turkey	EULEP5950-18
	Iran	IRANB315-08
	Kazakhstan	EULEP5956-18, EULEP5957-18, EULEP5958-18, EULEP5959-18, EULEP5960-18
	Russia	LEFIL147-10
I. feisthamelii	Morocco	EULEP5954-18, EULEP5955-18, EULEP5990-18, EULEP5996-18, EULEP5997-18, WMB245-11, WMB373-11, WMB399-11, WMB421-11, WMB3604-14, BDE293-19, BDE293-19, BDE294-19
	Algeria	EULEP5964-18, EULEP5981-18, EULEP6001-18, WMB1472-13, WMB1473-13, WMB1476-13
	Tunisia	EULEP5970-18, WMB1405-13, WMB1406-13, WMB6562-18
	Portugal	WMB4400-14, BDE553-19
	Spain	EULEP5949-18, 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
	France	EULEP002-14, 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).

Gene	Strand	Span		Anti-Codon	Start Codon		Stop Codon
		A	B		A	B	A	B
tRNA-Met	+	1–66	1–67	CAU	—	—	—	—
tRNA-Ile	+	67–130	68–131	GAU	—	—	—	—
tRNA-Gln	−	128–196	129–197	UUG	—	—	—	—
ND2	+	243–1256	256–1296	—	ATT	ATT	TAA	TAA
tRNA-Trp	+	1255–1319	1268–1332	UCA	—	—	—	—
tRNA-Cys	−	1312–1379	1325–1391	GCA	—	—	—	—
tRNA-Tyr	−	1380–1444	1394–1464	GUA	—	—	—	—
COX1	+	1445–2978	1468–2998	—	ATG	CGA 1	T-tRNA	T-tRNA
tRNA-Leu	+	2979–3045	2999–3065	UAA	—	—	—	—
COX2	+	3046–3727	3066–3747	—	ATG	ATG	T-tRNA	T-tRNA
tRNA-Lys	+	3728–3797	3748–3817	CUU	—	—	—	—
tRNA-Asp	+	3798–3865	3818–3884	GUC	—	—	—	—
ATP8	+	3866–4024	3885–4043	—	ATC	ATA	TAA	TAA
ATP6	+	4018–4695	4037–4714	—	ATG	ATG	TAA	TAA
COX3	+	4701–5492	4720–5508	—	ATA	ATA	TAA	TAA
tRNA-Gly	+	5496–5561	5512–5577	UCC	—	—	—	—
ND3	+	5562–5915	5578–5931	—	ATT	ATA	TAG	TAA
tRNA-Ala	+	5914–5976	5931–5993	UGC	—	—	—	—
tRNA-Arg	+	5977–6039	5994–6057	UCG	—	—	—	—
tRNA-Asn	+	6041–6105	6059–6123	GUU	—	—	—	—
tRNA-Ser	+	6107–6167	6125–6187	UCU	—	—	—	—
tRNA-Glu	+	6183–6247	6197–6263	UUC	—	—	—	—
tRNA-Phe	−	6246–6310	6262–6326	GAA	—	—	—	—
ND5	−	6311–8050	6327–8066	—	TTG	TTG	TAA	TAA
tRNA-His	−	8051–8116	8067–8132	GUG	—	—	—	—
ND4	−	8116–9455	8132–9471	—	ATG	ATG	T-tRNA	T-tRNA
ND4L	−	9456–9746	9472–9762	—	ATG	ATG	TAA	TAA
tRNA-Thr	+	9749–9813	9765–9828	UGU	—	—	—	—
tRNA-Pro	−	9814–9878	9829–9893	UGG	—	—	—	—
ND6	+	9881–10,411	9896–10,426	—	ATT	ATT	TAA	TAA
CYTB	+	10,416–11564	10,431–11,579	—	ATG	ATG	TAA	TAA
tRNA-Ser	+	11,580–11,647	11,597–11,665	UGA	—	—	—	—
ND1	−	11,665–12,603	11,683–12,621	—	ATG	ATG	TAG	TAG
tRNA-Leu	−	12,605–12,672	12,623–12,689	UAG	—	—	—	—
16S-rRNA	+	12,691–14,029	12,709–14,056	—	—	—	—	—
tRNA-Val	−	14,028–14,097	14,055–14,126	UAC	—	—	—	—
12S-rRNA	+	14,098–14,895	14,127–14,900	—	—	—	—	—

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.

	1	2a	2b	3
1. I. podalirius
2a. I. feisthamelii North Africa	2.234
2b. I. feisthamelii Iberia	0.134	2.184
3. I. podalirinus	5.309	5.530	5.280

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.

BIN Number	No. of Sequences	Species Included	Country/Region
BOLD:AAB2272	198	I. 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:AEA3098	25	I. feisthamelii	Morocco (13); Algeria (7); Tunisia (4); Unknown (1)

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.

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