International Scholarly Research Notices

International Scholarly Research Notices / 2013 / Article

Research Article | Open Access

Volume 2013 |Article ID 491636 | 14 pages |

Complete Mitogenomes of Euploea mulciber (Nymphalidae: Danainae) and Libythea celtis (Nymphalidae: Libytheinae) and Their Phylogenetic Implications

Academic Editor: B. Antonio
Received27 Nov 2012
Accepted13 Dec 2012
Published21 Feb 2013


The complete mitochondrial genome sequences of the two butterfly species Euploea mulciber (Lepidoptera: Nymphalidae: Danainae) and Libythea celtis (Lepidoptera: Nymphalidae: Libytheinae) were determined in this study, comprising 15,166 bp and 15,164 bp, respectively. The orientation and the gene order of the two mitogenomes are identical to those of most of the other lepidopteran species. All protein-coding genes of Euploea mulciber and Libythea celtis mitogenomes start with a typical ATN codon with the exception of COI gene which uses CGA as its initial codon. All tRNA genes possess the typical cloverleaf secondary structure except for tRNASer (AGN), which has a simple loop with the absence of the DHU stem. There are short microsatellite-like repeat regions, but no conspicuous macrorepeats scattered throughout the A + T-rich regions. Phylogenetic analysis among the available butterfly species suggests that Libythea celtis (Libytheinae) is closely related to Calinaga davidis (Calinaginae), indicating that the subfamily Libytheinae may not represent a basal lineage of the Nymphalidae as previously suggested, and that Euploea mulciber stands at the base of the nymphalid tree as a sister to all other nymphalids.

1. Introduction

The animal mitochondrial genome (mitogenome) is a circular molecule of 15–20 kb in length. It contains 37 conserved genes including 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 13 protein-coding genes (PCGs) involved in electron transport and oxidative phosphorylation [1, 2]. It also contains an A + T-rich region which is the largest noncoding area involved in the initiation and regulation of replication and transcription [3]. Mitogenome studies are important for comparative and evolutionary genomics, phylogenetics, molecular evolution, and population genetics due to the genome’s unique features, such as maternal inheritance, lack of extensive recombination, and accelerated nucleotide substitution rates [4, 5].

The Lepidoptera (butterflies and moths) is probably the largest insect order, containing over 165,000 described species. The systematics of the lepidopteran higher groups has long been a matter of contention [68]. In China, Chou’s taxonomic system [9] is widely adopted, in which the Chinese butterflies are split into 12 families and 32 subfamilies based on their morphological characteristics. However, another taxonomic system proposed by Wahlberg et al. (2005) [10], which is followed herein, has been commonly accepted by most butterfly researchers. In this system, the butterflies are classified into 2 superfamilies (Hesperioidea and Papilionoidea) and 5 families (Hesperiidae, Papilionidae, Pieridae, Lycaenidae, and Nymphalidae). Thus, the taxonomic levels and the phylogenetic relationships within and among butterfly lineages of some butterfly groups, such as the danainids and libytheines, remain controversial issues among different researchers.

Euploea mulciber is a member of the subfamily Danainae (formerly treated as a family). This medium-sized butterfly species is commonly called “striped blue crow” due to the male’s bright blue shot forewing and the female’s striped hindwing with narrow white streaks; its main host plants, Ficus and Nerium, are poisonous, mimicked by other butterfly species such as Elymnias malelas, Hypolimnas anomala, and Chilasa paradoxa. E. mulciber is distributed in India, Nepal, Bhutan, Burma, Malaysia, and China [9].

Libythea celtis is a species of the subfamily Libytheinae (formerly treated as a family). This species is recorded in Southern Europe, Northern Africa, and Asia, and their larvae generally feed on leaves of Celtis (Celtidaceae). The Libytheinae include only about 10 species in the world, so far as known, 3 of which are in China. They can be easily recognized by their unusually long labial palpus, although there are some other nymphalids that have an equally long or longer labial palpus [8]; the libytheine threadlike labial palpus looks like the petiole of a dead leaf, offering camouflage when the butterfly is taking a rest [11].

Currently, data of lepidopteran mitochondrial genomes are quite limited [1219]. Among these, 20 complete and 2 nearly complete mitochondrial sequences of butterflies have been determined. In this study, the complete mitogenomes of the two representative nymphalid species mentioned previously were determined and compared with other butterflies available, in order to provide more information for the phylogenetic studies of lepidopterans and further clarify their taxonomic status within the family Nymphalidae at the mitogenomic level.

2. Materials and Methods

2.1. Specimen Collection

Adult individuals of Euploea mulciber and Libythea celtis were collected in Dali, Yunnan province, and Huangshan, Anhui province, China, respectively. The fresh material was preserved in 100% ethanol and stored at −20°C before the DNA extraction.

2.2. DNA Extraction, PCR Amplification, and Sequencing

The whole genomic DNA was extracted from thoracic muscle tissue with the DNeasy Blood & Tissue kit (Qiagen) after Hao et al. (2005) [20]. Some universal PCR primers for short fragment amplifications of srRNA, COI, CytB, ND1, and COII genes were synthesized after Simon et al. (1994) [21], Caterino and Sperling (1999) [22], and Simmons and Weller (2001) [23]. The remaining primers for the two species were designed by the multiple sequence alignments of the complete mitogenomes of all the lepidopterans available, using ClustalX1.8 [24] and Primer Premier 5.0 softwares [25]. The entire mitogenomes of Euploea mulciber and Libythea celtis were amplified in five fragments (COI-COIII, COIII-ND4, ND4-CytB, and CytB-lrRNA, lrRNA-COI) and ten fragments (COI-COII, COII-COIII, COIII-ND5, ND5-ND4, ND4-CytB, CytB-ND1, ND1-lrRNA, lrRNA-srRNA, srRNA-ND2, and ND2-COI) using long PCR technique, which was performed using TaKaRa LA Taq polymerase with the following cycling parameters: 95°C for 5 minutes, 30 cycles of 95°C for 50 seconds, 47–51°C for 50 seconds, 68°C for 2 minutes and 30 seconds, and a final extension step of 68°C for 10 minutes. The PCR products were detected via electrophoresis in 1.2% agarose gel, purified using the 3S Spin PCR Product Purification Kit and sequenced directly with ABI-377 automatic DNA sequencer. All the amplified products were sequenced directly except the ND2-COI of Libythea celtis, which was sequenced after cloning. For each long PCR product, the full double-stranded sequence was determined by primer walking.

2.3. Sequence Analysis and Annotation

Raw sequence files were proofread and assembled with BioEdit version 7.0 [26]. PCGs and rRNAs were identified by sequence comparison using ClustalX1.8 software and the NCBI Internet BLAST search function [27]. tRNA gene analysis was conducted using tRNAscan-SE software v.1.21 [28], and the putative tRNAs, which were not found by tRNAscan-SE, were confirmed by sequence comparison of Euploea mulciber and Libythea celtis with other lepidopterans. Nucleotide composition and codon usage were calculated in MEGA 4.0 software [29]. The Euploea mulciber and Libythea celtis mitogenome sequence data have been deposited into the GenBank database under the accession numbers HQ378507 and HQ378508, respectively.

2.4. Phylogenetic Analysis

The multiple alignments of the concatenated 13 PCG nucleotide sequences of the 24 butterfly mitogenomes, including two newly determined and 22 available from GenBank (Table 1), were done by the ClustalX1.8 software and then manually proofread. The genetic distances were calculated based on selected sequence evolutionary models; meanwhile, the neighbor joining (NJ) [30] and Bayesian inference (BI) [31] phylogenetic trees were reconstructed based on the PCG sequences using one moth species, Lymantria dispar (GenBank accession no. FJ617240) as the outgroup. In the NJ analysis, the Kimura 2 parameter (K2P) evolutionary model [32] was selected, and the bootstrap values with internal branch tests were obtained by 1,000 replicates to estimate the support levels for the nodes in the resultant topologies using the MEGA 4.0 software. The Bayesian analyses were performed based on the PCG nucleotide sequences using MrBayes 3.1.2 [33, 34] under the model GTR+I+G chosen by the modeltest 3.06 software [35], and the Markov’s chains were run simultaneously for 1,000,000 generations sampled every 100 generations; the first 2,500 trees were discarded as burn-in samples, and the remaining trees were used to generate a majority rule consensus tree, in which the percentage of samples recovering a particular clade represents its support measured as posterior probabilities.

Whole genomePCGblrRNAsrRNAA + T-rich region
Family/subfamilySpeciesSizeA + TNo. codonsaA + TSizeA + TSizeA + TSizeA + TGen of Bank accession no.References

Papilionidae/PapilioninaeTroides aeacus 15,26380.2374579.11234 83.3784 85.0419 89.8EU625344 Unpublished
Papilio xuthus 13,96480.0358378.91327 83.6598 84.5EF176224[63]
Agehana maraho 16,09480.5371879.21333 83.7779 85.51270 95.0FJ810212[49]
Teinopalpus aureus 15,24279.8372078.31320 82.4781 85.6395 93.1HM563681[64]
Parnassius bremeri 15,38981.3372380.21344 83.9773 85.1504 93.6FJ871125[13]
Papilionidae/ParnassiinaeSericinus montela 15,24280.8369180.21338 83.6759 84.6408 94.1HQ259122[18]
Luehdorfia chinensis 13,86080.5355179.6134284.046081.5EU622524 Unpublished
Hesperiidae/PyrginaeCtenoptilum vasava 15,46880.5369878.91343 84.1774 86.442988.1JF713818[19]
Pieridae/PierinaeArtogeia melete 15,14079.8371578.41319 83.4777 85.5351 89.2NC_010568[14]
Pieris rapae 15,15779.7372278.31320 84.0764 85.039391.6HM156697[39]
Lycaenidae/TheclinaeCoreana raphaelis 15,31482.7370881.51330 85.3777 85.8375 94.1DQ102703[12]
Protantigius superans 15,24881.7371280.31331 85.1739 85.6361 93.6HQ184265[65]
Lycaenidae/AphnaeinaeSpindasis takanonis 15,349 82.4371981.01333 85.6777 84.7371 94.6HQ184266[65]
Apatura ilia 15,24280.5371178.91333 84.077684.940392.5JF439725[50]
Nymphalidae/ApaturinaeSasakia charonda 15,24479.9369578.21323 84.4775 85.0380 91.8NC_014224Unpublished
Sasakia  15,22279.9358678.31311 84.277585.038091.8NC_014223Unpublished
Nymphalidae/NymphalinaeKallima inachus 15,18380.3372179.2133582.7774 85.1376 92.0JN857943[66]
Nymphalidae/HeliconiinaeArgyreus hyperbius 15,15680.8371879.51330 84.5778 85.2349 95.4JF439070[37]
Acraea issoria 15,24579.7371778.0133183.9788 83.7430 96.0GQ376195[15]
Nymphalidae/LimenitidinaeParathyma sulpitia 15,26881.9372980.61319 84.7779 85.7349 94.6JQ347260[17]
Nymphalidae/CalinaginaeCalinaga davidis 15,26780.4373778.91337 83.8773 85.9389 92.0NC_015480[67]
Nymphalidae/DanainaeEumenis autonoe 15,489 79.1372876.81335 83.7775 85.3678 94.5GQ868707[68]
Nymphalidae/LibytheinaeLibythea celtis 15,16481.2372280.01335 84.777585.432896.3HQ378508This study
Nymphalidae/DanainaeEuploea mulciber 15,16681.5370580.2131484.677685.339993.5HQ37507This study
LymantriidaeLymantria dispar 15,569 79.93742 77.8 1351 84.2799 85.2435 96.1NC_012893[69]

aTermination codons were excluded in total codon count. bProtein coding genes. The bar (—) indicates lack of sequence information on the A + T-rich region in the genome. Sasakia charonda kuriyamaensis.

3. Results and Discussion

3.1. Genome Organization and Structure

The mitogenomes of Euploea mulciber and Libythea celtis both consist of 13 PCGs, two ribosomal RNA genes for the small and large subunits (srRNA and lrRNA), and 22 tRNAs, and an A + T-rich region (see Tables 1 and 2 and Figure 1). Like many other insect mitogenomes, its major strand code for more genes (9PCGs and 14 tRNAs), whereas less genes were coded in the minor strand (4 PCGs, 8 tRNAs, and 2 rRNA genes). The mitogenome size of 15,166 bp and 15,164 bp determined here is close to most lepidopteran mitogenomes although slightly shorter than those of most completely sequenced lepidopteran insects (Table 1). The Euploea mulciber mitochondrial genes are interleaved with a total of 117 bp intergenic spacer sequences, which are spread over 13 regions, ranging from 1 to 46 bp, and only three spacers span longer than 10 bp, located between and ND2 (46 bp), between ND5 and (15 bp), and between (UCN) and ND1 (20 bp). Additionally, a total of 32 bp overlapped nucleotides are spread over 12 regions, and every region is shorter than 10 bp in length. The Libythea celtis mitochondrial genes are interleaved with a total of 97 bp intergenic spacer sequences, which are also spread over 13 regions, ranging from 1 to 52 bp, among which only two spacers span longer than 10 bp, located between and ND2 (52 bp) and between (UCN) and ND1 (17 bp). Besides, a total of 27 bp overlapped nucleotides are spread over 9 regions, and every region is shorter than 10 bp in size. The nucleotide compositions of A + T in Euploea mulciber and Libythea celtis mitogenomes are 81.5% and 81.2%, respectively, showing strong A + T bias. Correspondingly, the AT contents of the two butterfly species are up to 80.2% and 80.0% for the PCGs, 84.6% and 84.7% for the lrRNA gene, 85.3% and 85.4% for the srRNA gene, and 93.5% and 96.3% for the control region. This case is similar to those observed in other rhopaloceran mitogenomes, ranging from 79.7% in Pieris rapae and Acraea issoria to 82.7% in Coreana raphaelis (Tables 1 and 2).

GeneDirectionNucleotide no.Size (bp) Intergenic nucleotidesAnticodonStart codonStop codon

tRNAMetFF1–681–68686832–34 CAT32–34 CAT
tRNAIle(AUR)FF78–14067–1316365 9 −2106–108 GAT95–97 GAT
tRNAGlnRR138–206129–1976969−3−3174–176 TTG165–167 TTG
ND2FF253–1251250–12639991014 46 52 ATTATCTAATAA
tRNATrpFF1250–13171265–13316867−2 1 1281–1283 TCA1296–1298 TCA
tRNACysRR1310–13721324–13866363−8−81340–1342 GCA1355–1357 GCA
tRNATyrRR1374–14391387–14516665 1 0 1406–1408 GTA1418–1420 GTA
COIFF1442–29721458–298815311531 2 6 CGACGAT-tRNALeu(UUR)T-tRNALeu(UUR)
tRNALeu(UUR)FF2973–30392989–30556767 0 0 3003–3005 TAA3019–3021 TAA
COIIFF3040–37123056–3737673682 0 0 ATGATGT-tRNALysT-tRNALys
tRNALysFF3713–37833738–38087171 0 0 3743–3745 CTT3768–3770 CTT
tRNAAspFF3783–38483808–38736666−1−13813–3815 GTC3838–3840 GTC
ATP8FF3849–40103874–4041162168 0 0 ATTATTTAATAA
tRNAGlyFF5476–55415503–55696667 6 2 5506–5508 TCC5533–5535 TCC
ND3FF5542–58955570–5923354354 0 0 ATTATTTAGTAA
tRNAAlaFF5894–59595928–59966669−2 4 5925–5927 TGC5961–5963 TGC
tRNAArgFF5959–60245997–60596663−1 0 5987–5989 TCG6024–6026 TCG
tRNAAsnFF6026–60916065–61306666 1 5 6057–6059 GTT6096–6098 GTT
tRNASer(AGN)FF6090–61496129–61906062−2−26110–6112 TCT6150–6152 GCT
tRNAGluFF6158–62236192–62596668 8 1 6188–6190 TTC6224–6226 TTC
tRNAPheRR6222–62866258–63256568−2−26254–6256 GAA6290–6292 GAA
ND5RR6287–80036326–805717171732 0 0 ATTATTT-tRNAPheT-tRNAPhe
tRNAHisRR8019–80838058–81226565 15 0 8051–8053 GTG8090–8092 GTG
ND4RR8084–94228123–946113391339 0 0 ATGATGT-tRNAHisT-tRNAHis
ND4LRR9422–97099464–9754288291−1 2 ATGATGTAATAG
tRNAThrFF9712–97759757–98216465 2 2 9742–9744 TGT9788–9790 TGT
tRNAProRR9776–98399822–98866465 0 0 9808–9810 TGG9854–9856 TGG
ND6FF9842–103669889–10419525531 2 2 ATAATTTAATAA
CytBFF10371–11,51910419–11,56711491149 4 −1ATGATGTAATAA
tRNASer(UCN)FF11,518–11,58211,570–11,6366567−2 2 11547–11549 TGA11600–11602 TGA
ND1RR11,603–12,54411,654–12,592942939 20 17 ATGATGTAATAA
tRNALeu(CUN)RR12,546–12,61212,594–12,6606767 1 1 12581–12583 TAG12629–12631 TAG
lrRNARR12,613–13,92612,661–13,99513141335 0 0
tRNAValRR13,927–13,99113,996–14,0616566 0 0 13961–13963 TAC14029–14031 TAC
srRNARR13,992–14,76714,062–14,836776775 0 0
D-loopFF14,768–15,16614,837–15,164399328 0 0

1* indicates the species of Euploea mulciber and 2* indicates the species of Libythea celtis.
3.2. Protein-Coding Genes

The total codon numbers of the 13 PCGs for Euploea mulciber and Libythea celtis are 3722 and 3705, respectively (Table 1). The nucleotide composition for each codon position and the codon usage of PCGs are shown in Tables 3 and 4. For codon usage, some codons have much more numbers, such as the TTA(L), ATT(I), TTT(F), and ATA(M), while some codons, such as CGT(L), AGG(S), and GGC(G) are not used. In addition, the synonymous codon bias, namely, the relative synonymous codon usage (RSCU) are detected, and their bias patterns roughly correspond to those of codon usage, for example, the four most frequently used codons (TTA, ATT, TTT, and ATA) account for 10.14% of all the codons. The AT and GC nucleotide skewness values for the PCGs as well as the whole genomes are listed in Table 5, and the results showed that they are slightly negative and positive for the whole PCGs and their minor strand of the two nymphalid species, respectively, indicating that more T and G are used than A and C; however, both values are slightly negative for the major strands, indicating that more T and C are used in these coding regions. In general, these codon constitutional and biased patterns are significantly congruent with those of other lepidopteran species determined to date [13, 18, 19, 3638].

Species1st codon position2nd codon position3rd codon positionOverall

Papilio xuthus 36.937.210.515.421.948.916.213.040.648.96.53.933.
Agehana maraho 36.937.210.515.421.948.916.213.040.648.96.53.933.
Troides aeacus 36.637.010.316.122.548.415.913.240.552.24.92.433.245.810.410.6
Sericinus montela 37.537.89.615.122.048.916.
Teinopalpus aureus
Luehdorfia chinensis 36.637.410.115.922.248.516.213.142.751.33.72.333.845.710.010.4
Parnassius bremeri
Coreana raphaelis 38.338.18.814.922.049.015.813.245.851.
Spindasis takanonis 38.638.
Protantigius superans 37.737.79.215.322.048.815.913.
Pieris rapae
Artogeia melete 36.736.810.316.221.947.916.813.442.349.84.63.433.644.810.611.0
Apatura ilia
Sasakia charonda 36.737.69.815.922.348.316.113.340.948.56.14.433.344.810.711.2
Sasakia 36.837.79.715.822.248.516.113.240.648.86.04.633.245.010.611.2
Kallima inachus 36.437.210.116.321.848.516.413.342.551.23.52.733.645.610.010.8
Argyreus hyperbius 37.437.19.915.622.648.
Acraea issoria 36.636.710.715.923.047.816.213.139.750.16.43.733.144.911.110.9
Parathyma sulpitia 36.937.99.815.422.348.216.413.
Calinaga davidis 36.337.710.415.722.248.416.413.140.751.55.12.733.045.810.610.5
Eumenis autonoe 35.936.311.316.421.548.416.413.739.748.87.14.432.344.511.611.5
Euploea mulciber 37.437.49.515.721.948.516.513.
Libythea celtis 36.637.69.616.
Ctenoptilum vasava

Start codons and stop codons were excluded in the count. Sasakia charonda kuriyamaensis.

Codon (RSCU)

UUU(F)360 (1.88)/346 (1.89)
UUC(F)23 (0.12)/21 (0.11)
UUA(L)489 (5.37)/483 (5.35)
UUG(L)5 (0.05)/8 (0.09)
CUU(L)35 (0.38)/34 (0.38)
CUC(L)1 (0.01)/3 (0.03)
CUA(L)16 (0.18)/14 (0.15)
CUG(L)0 (0)/0 (0)
AUU(I)425 (1.95)/436 (1.91)
AUC(I)11 (0.05)/21 (0.09)
AUA(M)276 (1.94)/287 (1.93)
AUG(M)9 (0.06)/10 (0.07)
GUU(V)69 (1.99)/63 (1.98)
GUC(V)2 (0.06)/1 (0.03)
GUA(V)65 (1.87)/62 (1.95)
GUG(V)3 (0.09)/1 (0.03)
UCU(S)120 (2.94)/97 (2.45)
UCC(S)7 (0.17)/8 (0.2)
UCA(S)83 (2.03)/94 (2.37)
UCG(S)1 (0.02)/5 (0.13)
CCU(P)73 (2.39)/77 (2.52)
CCC(P)9 (0.3)/10 (0.33)
CCA(P)39 (1.28)/35 (1.15)
CCG(P)1 (0.03)/0 (0)
ACU(T)84 (2.15)/92 (2.36)
ACC(T)2 (0.05)/7 (0.18)
ACA(T)70 (1.79)/53 (1.36)
ACG(T)0 (0)/4 (0.1)
GCU(A)70 (2.28)/75 (2.36)
GCC(A)6 (0.2)/1 (0.03)
GCA(A)46 (1.5)/49 (1.54)
GCG(A)1 (0.03)/2 (0.06)
UAU(Y)173 (1.9)/18 (1.94)
UAC(Y)9 (0.1)/6 (0.06)
UAA(*)0 (0)/0 (0)
UAG(*)0 (0)/0 (0)
CAU(H)59 (1.79)/60 (1.79)
CAC(H)7 (0.21)/7 (0.21)
CAA(Q)61 (1.91)/56 (1.9)
CAG(Q)3 (0.09)/3 (0.1)
AAU(N)250 (1.91)/24 (1.91)
AAC(N)12 (0.09)/11 (0.09)
AAA(K)93 (1.81)/99 (1.96)
AAG(K)10 (0.19)/2 (0.04)
GAU(D)69 (1.97)/58 (1.9)
GAC(D)1 (0.03)/3 (0.1)
GAA(E)60 (1.76)/68 (1.89)
GAG(E)8 (0.24)/4 (0.11)
UGU(C)26 (1.73)/28 (1.65)
UGC(C)4 (0.27)/6 (0.35)
UGA(W)93 (1.96)/92 (1.98)
UGG(W)2 (0.04)/1 (0.02)
CGU(R)10 (0.77)/14 (1.1)
CGC(R)1 (0.08)/1 (0.08)
CGA(R)39 (3)/36 (2.82)
CGG(R)2 (0.15)/0 (0)
AGU(S)32 (0.78)/29 (0.73)
AGC(S)3 (0.07)/2 (0.05)
AGA(S)81 (1.98)/81 (2.04)
AGG(S)0 (0)/1 (0.03)
GGU(G)50 (1)/55 (1.13)
GGC(G)0 (0)/2 (0.04)
GGA(G)126 (2.52)/120 (2.47)
GGG(G)24 (0.48)/17 (0.35)

: frequency of codon used; RSCU: relative synonymous codon usage; *stop codon.
Start codons and stop codons were excluded in total codon counts.

Major strand PCGsMinor strand PCGsWhole PCGsWhole genome
SpeciesA + TAT skewGC skewA + TAT skewGC skewA + TAT skewGC skewA + TAT skewGC skew

Papilio xuthus 77.1 79.8 0.334 78.1 80.0
Agehana maraho 77.1 79.8 0.334 78.1 80.6
Teinopalpus aureus 77.2 80.0 0.322 78.3 79.8
Troides aeacus 77.7 81.1 0.332 79.1 80.2
Parnassius bremeri 78.9 82.2 0.281 80.2 81.3 −0.011−0.194
Sericinus montela 78.5 81.8 0.301 79.8 81.0
Luehdorfia chinensis 78.0 81.9 0.296 79.6 80.5
Ctenoptilum vasava 77.2 81.6 0.285 78.9 80.5
Pieris rapae 77.2 79.8 0.358 78.2 79.7
Artogeia melete 77.7 79.7 0.324 78.4 79.8 0.012 −0.222
Coreana raphaelis 80.3 83.4 0.270 81.5 82.7
Protantigius superans 79.3 81.9 0.276 80.3 81.7
Spindasis takanonis 80.1 82.7 0.338 81.1 82.4
Apatura ilia 77.9 80.7 0.314 79.0 80.4
Sasakia charonda 76.9−0.118−0.152 80.0 −0.194 0.330 78.1 −0.147 0.023 79.9 −0.006−0.219
Sasakia  77.2 79.7 0.324 78.2 79.9
Kallima inachus 78.1 81.1 0.336 79.2 80.3
Parathyma sulpitia 79.2−0.172−0.100 83.1 −0.154 0.266 80.6 −0.164 0.026 81.9 −0.048−0.178
Eumenis autonoe 75.4−0.135−0.187 79.3 −0.193 0.337 76.8 −0.159−0.004 79.1 −0.016−0.244
Calinaga davidis 77.5−0.164−0.147 81.1 −0.159 0.270 78.8 −0.162−0.005 80.4 −0.045−0.200
Acraea issoria 76.7−0.142−0.176 80.1 −0.164 0.307 78.0 −0.146−0.009 79.7 −0.024−0.238
Argyreus hyperbius 77.8−0.136−0.153 82.0 −0.166 0.322 79.4 −0.149 0.010 80.8 −0.025−0.219
Libythea celtis 78.8
Euploea mulciber 79.0

Start codons and stop codons were excluded in the count.
The skewness of whole PCGs and whole genome were calculated from major strands. Sasakia charonda kuriyamaensis.

Except for COI, 12 out of 13 PCGs in Euploea mulciber and Libythea celtis use standard ATN start codons: ATA for ND6; ATT for ND2, ATP8, ND3, and ND5; ATG for COII, ATP6, COIII, ND4, ND4L, CytB, and ND1 in Euploea mulciber; ATC for ND2; ATT for ATP8, ND3, ND5, and ND6; and ATG for COII, ATP6, COIII, ND4, ND4L, CytB, and ND1 in Libythea celtis.

In Figure 2, we show the alignment of the initiation site for the COI genes of 24 completely sequenced rhopaloceran mitogenomes. All the mitogenomes harbor a few canonical ATN initiators detected in the start region of the COI gene or in the immediately neighboring sequence of the precedent . However, in eleven of them (Artogeia melete, Coreana raphaelis, Argyreus hyperbius, Teinopalpus aureus, Papilio maraho, Luehdorfia chinensis, Parnassius bremeri, Protantigius superans, Troides aeacus, Libythea celtis, and Euploea mulciber), a TAG (stop codon) is present at the beginning region of the COI gene, while in the other five mitogenomes (Papilio xuthus, Sericinus montela, Ctenoptilum vasava, and two Sasakia subspecies), the proposed ATN initiators are present within the region coding for . Thus, the ATN initiators should not be considered as the start codon for the COI gene. According to these criteria, the first nonoverlapping codon in the COI gene is the CGA designating arginine existing in a highly conserved region across rhopaloceran insects [13].

Nine of the PCGs in Euploea mulciber and Libythea celtis harbor the complete termination codon TAA or TAG, that is, eight protein-coding sequences stop with TAA, one with TAG. The other four are with the incomplete termination codon T. This phenomenon of partial termination codons (i.e., T or TA) is observed in all sequenced lepidopteran insects and has been interpreted in terms of posttranscriptional polyadenylation, by which “A” residue(s) are added to create TAA terminator [13].

3.3. Ribosomal RNAs and Transfer RNAs

The lrRNA genes of the Euploea mulciber and Libythea celtis mitogenomes are 1314 and 1335 bp in length, respectively. As has been observed in other insects, this gene is located between (CUN) and genes [12, 13, 15, 16, 19, 37, 3945]. In the same way, the srRNA genes of the Euploea mulciber and Libythea celtis mitogenomes are 776 and 775 bp in length, respectively, and located between and A + T-rich region. Both of the two rRNA genes are encoded by the minor strand, and the lengths of them are well within the size range of other insects determined. The AT contents of lrRNA and srRNA genes are 84.6% and 85.3% in Euploea mulciber and 84.7% and 85.4% in Libythea celtis, respectively, and these values are also well within the range of the sequenced insects.

Like other insects, both Euploea mulciber and Libythea celtis harbor 22 tRNAs, among which both Leucine and Serine have two tRNAs, and the left tRNAs for each of 18 amino acid residues are all one in number (Figure 1). In this study, all the tRNAs of Euploea mulciber and Libythea celtis evidence the typical cloverleaf structures with the exception of (AGN), whose dihydrouridine (DHU) arm forms a simple loop (maps of secondary structure are not shown here). The case of (AGN) with the simple loop in the DHU arm has also been detected in most of insect species including the lepidopterans determined till now [1219, 36, 37, 39, 42, 4548].

3.4. A + T-Rich Region

The A + T-rich region is known for the initiation of replication in vertebrates and invertebrates and thus also called as the control region. The Euploea mulciber and Libythea celtis A + T-rich regions located between srRNA and were 399 and 328 bp in length, respectively (see Figure 1 and Table 2). Though the A + T-rich region of Libythea celtis is the shortest, its A + T content is the highest (96.3%) in all of the completely sequenced rhopaloceran mitogenomes known to date. In general, this region harbors the highest A + T content in the whole regions of the insect mitogenomes, and this feature is well presented in those of the two nymphalid species in this study. However, the regions of these two species harbor no large sequence repeats detected frequently in other insect groups, including some of the lepidopterans. Nonetheless, there are several short sequence repeats scattered throughout the whole region, for example, a poly T closed to the srRNA, a TA repeat in the middle of the A + T-rich region and a poly A closed to .

Alignment of the initiation sites for the A + T-rich regions of 22 completely sequenced rhopaloceran mitogenomes is shown in Figure 3, and the results showed that all the A + T-rich regions harbor a poly-T stretch preceded by the ATAGA motif. The Euploea mulciber and Libythea celtis harbored a 21 bp and 19 bp long poly-T stretch, respectively. To the best of our knowledge, the poly-T stretch is quite conserved in all sequenced rhopaloceran insects, ranging from 12 bp in Agehana maraho [49] to 21 bp in Euploea mulciber of this study and Apatura ilia [50] (Figure 3), and it has been suggested as a possible recognition site for the replication initiation of the mtDNA minor strand owing to the facts that the insect mitochondrial A + T-rich region has been revealed to harbor the replication originating sites for both strands in Drosophila [40, 41], and its neighboring region has been identified as the position of the minor-strand replication origin in Bombyx mori [51].

A microsatellite-like elements (AT)3(TA)6 and (TA)12 preceded by the ATTTA motif are presented in the middle of the Euploea mulciber and Libythea celtis A + T-rich region, respectively. Similar cases are commonly found in all other sequenced lepidopteran species [1215, 17, 19, 3639, 4244, 4648]. Additionally, a 12 bp poly A interrupted by T and a 7 bp poly A interrupted by G presented immediately the upstream of the . This poly A element at the end of the A + T-rich region and the short repeating sequences are probably universal features in lepidopterans, and their unknown functions await further in-depth studies.

3.5. Phylogenetic Analysis

Thirteen concatenated PCGs nucleotide sequences of complete rhopaloceran mitogenomes were used to analyze the phylogenetic relationships among the butterfly groups in this study. The resultant 14-taxon (including outgroup) data set is 11,459 characters after removal of gaps, among which 5402 are variable and 3759 are parsimony-informative. The results of neighbor-joining (NJ) and Bayesian inference (BI) analyses show that the topologies of the NJ and BI trees shown in Figure 4 are identical only with minor differences in their node support values. The trees indicates the following relationship: (Papilionidae + (Pieridae + (Nymphalidae + Lycaenidae))), which is congruent with that obtained by Walhberg et al. (2005): (Hesperiidae + (Papilionidae + (Pieridae + (Nymphalidae + (Lycaenidae + Riodinidae))))) [10], though the data from the Hesperiidae and Riodinidae is not available here. Additionally, within the Nymphalidae, Euploea mulciber stands at the base of the nymphalid tree, as a sister to all the remaining groups of the family, and Libythea celtis (Libytheinae) is the nearest sister tothe Calinaga davidis (Calinaginae).

The danaids, formerly classified as familial taxa under the family Nymphalidae, have now been relegated to the subfamily Danainae. They possess unique features, such as the brush-like modified forelegs, poisonousness to their potential predators, which are markedly distinct with other nymphalid butterflies, and have been proposed as one of the basal groups in the family Nymphalidae by Walhberg et al. (2003) based on molecular evidences [52]. Morphologically, Freitas and Brown (2004) also revealed that the danaid clade (including Danaini, Tellervini, and Ithomiini) appeared as the basal group of the Nymphalidae excluding Libytheinae based on the immature features [53]. Hao et al. (2007) reconstructed the mitochondrial lrRNA secondary structures of the main butterfly lineages, and the results showed that the morphological characteristics of the danaid species Euploea core and Parantica aglea were markedly different from other nymphalid groups [54]. In this study, both NJ and BI trees indicate that Euploea mulciber is sister to all other nymphalid species, standing at the base of the whole nymphalid tree. Therefore, we conclude that the danaid butterflies are the most primitive group of Nymphalidae.

The Libytheinae is one of the most morphologically unusual groups of butterflies, and their relationship with other butterfly groups remains controversial. Traditionally, they are considered as the basal group of Nymphalidae [16, 5557]. Some scholars even considered them as a separate family owing to their unique morphological characters such as the markedly prolonged snout and well-developed foreleg in females [58, 59]. In addition, some phylogenetic analyses based on morphological, molecular, or combined data also proposed that Libytheinae is the basal lineage of the Nymphalidae [10, 6062]. However, this study reveals that Libythea celtis is closely related to Calinaga davidis (Calinaginae) with a weak bootstrap support value (65%) in NJ tree, whereas, stands as a sister to the species grouping of Limenitidinae, Heliconiinae, Nymphalinae, and Apaturinae with a high posterior probability value (0.97) in BI tree (Figure 4). Thus, its phylogenetic position within Nymphalidae awaits further clarification in the future, on the grounds of more taxa samplings as well as more realistic analysis methods. In conclusion, it is obvious that the Danainae is the basal branch of the Nymphalidae, rather than the Libytheinae.


This work was supported by the National Science Foundation of China (Grant no. 41172004), the CAS/SAFEA International Partnership Program for Creative Research Teams, Chinese Academy of Sciences (Grant no. KZCX22YW2JC104), the Provincial Key Projects of Natural Science Foundation, Colleges of Anhui Province (Grant no. KJ2010A142), and the Opening Funds from the State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences (Grant no. 104143).


  1. D. R. Wolstenholme, “Animal mitochondrial DNA: structure and evolution,” International Review of Cytology, vol. 141, pp. 173–216, 1992. View at: Google Scholar
  2. J. L. Boore, “Animal mitochondrial genomes,” Nucleic Acids Research, vol. 27, no. 8, pp. 1767–1780, 1999. View at: Publisher Site | Google Scholar
  3. D. A. Clayton, “Transcription and replication of animal mitochondrial DNAs,” International Review of Cytology, vol. 141, pp. 217–232, 1992. View at: Google Scholar
  4. W. M. Brown, “Evolution of animal mitochondrial DNA,” in Evolution of Genes and Proteins, M. Nei and P. K. Koehn, Eds., pp. 62–88, Sinauer, Sunderland, UK, 1983. View at: Google Scholar
  5. J. C. Avise, Molecular Markers, Natural History and Evolution, Champman & Hall, New York, NY, USA, 1994.
  6. P. R. Ackery, “Systematic and faunistic studies on butterflies,” in The Biology of Butterflies, R. I. Vane-Wright and P. R. Ackery, Eds., pp. 9–21, Academic Press, London, UK, 1984. View at: Google Scholar
  7. P. R. Ackery, R. de Jong, and R. I. Vane-Wright, “The butterflies: hedyloidea, Hesperioidea and Papilionoidae,” in Lepidoptera: Moths and Butterflies. 1. Evolution, Systematics and Biogeography, Handbook of Zoology, N. P. Kristensen, Ed., vol. 4, pp. 264–300, De Gruyter, Berlin, Germany, 1999. View at: Google Scholar
  8. D. J. Harvey, “Higher classification of the Nymphalidae, Appendix B,” in The Development and Evolution of Butterfly Wing Patterns, H. F. Nijhout, Ed., pp. 255–273, Smithsonian Institution Press, Washington, DC, USA, 1991. View at: Google Scholar
  9. I. O. Chou, Classification and Identification of Chinese Butterflies, Henan Scientific and Technological Publishing House, Zhengzhou, China, 1998.
  10. N. Wahlberg, M. F. Braby, A. V. Z. Brower et al., “Synergistic effects of combining morphological and molecular data in resolving the phylogeny of butterflies and skippers,” Proceedings of the Royal Society B, vol. 272, no. 1572, pp. 1577–1586, 2005. View at: Publisher Site | Google Scholar
  11. A. Y. Kawahara, “Behavioral observations of Libytheana carinenta Cramer,” News of the Lepidopterists' Society, vol. 45, pp. 107–108, 2003. View at: Google Scholar
  12. I. Kim, E. M. Lee, K. Y. Seol et al., “The mitochondrial genome of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae),” Insect Molecular Biology, vol. 15, no. 2, pp. 217–225, 2006. View at: Publisher Site | Google Scholar
  13. M. I. Kim, J. Y. Baek, M. J. Kim et al., “Complete nucleotide sequence and organization of the mitogenome of the red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) and comparison with other lepidopteran insects,” Molecules and Cells, vol. 28, no. 4, pp. 347–363, 2009. View at: Publisher Site | Google Scholar
  14. G. Hong, S. Jiang, M. Yu et al., “The complete nucleotide sequence of the mitochondrial genome of the cabbage butterfly, Artogeia melete (Lepidoptera: Pieridae),” Acta Biochimica et Biophysica Sinica, vol. 41, no. 6, pp. 446–455, 2009. View at: Publisher Site | Google Scholar
  15. J. Hu, D. Zhang, J. Hao, D. Huang, S. Cameron, and C. Zhu, “The complete mitochondrial genome of the yellow coaster, Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini): sequence, gene organization and a unique tRNA translocation event,” Molecular Biology Reports, vol. 37, no. 7, pp. 3431–3438, 2010. View at: Publisher Site | Google Scholar
  16. Y. J. Gong, B. C. Shi, Z. J. Kang, F. Zhang, and S. J. Wei, “The complete mitochondrial genome of the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae),” Molecular Biology Reports, pp. 1–8, 2011. View at: Publisher Site | Google Scholar
  17. L. L. Tian, X. Y. Sun, M. Chen, Y. H. Gai, J. S. Hao, and Q. Yang, “Complete mitochondrial genome of the five-dot sergeant Parathyma sulpitia (Nymphalidae: Limenitidinae) and its phylogenetic implications,” Zoology Research, vol. 33, no. 2, pp. 133–143, 2012. View at: Google Scholar
  18. L. W. Ji, J. S. Hao, Y. Wang, D. Y. Huang, J. L. Zhao, and C. D. Zhu, “The complete mitochondrial genome of the dragon swallowtail, Sericinus montela Gray (Lepidoptera: Papilionidae) and its phylogenetic implication,” Acta Entomologica Sinica, vol. 55, no. 1, pp. 91–100, 2012. View at: Google Scholar
  19. J. S. Hao, Q. Q. Sun, H. B. Zhao, X. Y. Sun, Y. H. Gai, and Q. Yang, “The complete mitochondrial genome of Ctenoptilum vasava (Lepidoptera: Hesperiidae: Pyrginae) and its phylogenetic implication,” Comparative and Functional Genomics, vol. 2012, Article ID 328049, 13 pages, 2012. View at: Publisher Site | Google Scholar
  20. J. S. Hao, C. X. Li, X. Y. Sun, and Q. Yang, “Phylogeny and divergence time estimation of cheilostome bryozoans based on mitochodrial 16S rRNA sequences,” Chinese Science Bulletin, vol. 12, pp. 1205–1211, 2005. View at: Google Scholar
  21. C. Simon, F. Frati, A. Beckenbach, B. Crespi, H. Liu, and P. Flook, “Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers,” Annals of the Entomological Society of America, vol. 87, no. 6, pp. 651–701, 1994. View at: Google Scholar
  22. M. S. Caterino and F. A. H. Sperling, “PapilioPhylogeny based on mitochondrial cytochrome oxidase I and II genes,” Molecular Phylogenetics and Evolution, vol. 11, no. 1, pp. 122–137, 1999. View at: Publisher Site | Google Scholar
  23. R. B. Simmons and S. J. Weller, “Utility and evolution of cytochrome b in insects,” Molecular Phylogenetics and Evolution, vol. 20, no. 2, pp. 196–210, 2001. View at: Publisher Site | Google Scholar
  24. J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins, “The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools,” Nucleic Acids Research, vol. 25, no. 24, pp. 4876–4882, 1997. View at: Publisher Site | Google Scholar
  25. V. K. Singh, A. K. Mangalam, S. Dwivedi, and S. Naik, “Primer premier: program for design of degenerate primers from a protein sequence,” BioTechniques, vol. 24, no. 2, pp. 318–319, 1998. View at: Google Scholar
  26. T. A. Hall, “BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT,” Nucleic Acids Symposium Series, no. 41, pp. 95–98, 1999. View at: Google Scholar
  27. S. F. Altschul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, “Basic local alignment search tool,” Journal of Molecular Biology, vol. 215, no. 3, pp. 403–410, 1990. View at: Publisher Site | Google Scholar
  28. T. M. Lowe and S. R. Eddy, “tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence,” Nucleic Acids Research, vol. 25, no. 5, pp. 955–964, 1997. View at: Google Scholar
  29. S. Kumar, K. Tamura, and M. Nei, “MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment,” Briefings in Bioinformatics, vol. 5, no. 2, pp. 150–163, 2004. View at: Google Scholar
  30. N. Saitou and M. Nei, “The neighbor-joining method: a new method for reconstructing phylogenetic trees,” Molecular Biology and Evolution, vol. 4, no. 4, pp. 406–425, 1987. View at: Google Scholar
  31. Z. Yang and B. Rannala, “Bayesian phylogenetic inference using DNA sequences: a Markov Chain Monte Carlo method,” Molecular Biology and Evolution, vol. 14, no. 7, pp. 717–724, 1997. View at: Google Scholar
  32. M. Kimura, “A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences,” Journal of Molecular Evolution, vol. 16, no. 2, pp. 111–120, 1980. View at: Google Scholar
  33. J. P. Huelsenbeck and F. Ronquist, “MRBAYES: bayesian inference of phylogenetic trees,” Bioinformatics, vol. 17, no. 8, pp. 754–755, 2001. View at: Google Scholar
  34. F. Ronquist and J. P. Huelsenbeck, “MrBayes 3: bayesian phylogenetic inference under mixed models,” Bioinformatics, vol. 19, no. 12, pp. 1572–1574, 2003. View at: Publisher Site | Google Scholar
  35. D. Posada and K. A. Crandall, “MODELTEST: testing the model of DNA substitution,” Bioinformatics, vol. 14, no. 9, pp. 817–818, 1998. View at: Google Scholar
  36. P. Salvato, M. Simonato, A. Battisti, and E. Negrisolo, “The complete mitochondrial genome of the bag-shelter moth Ochrogaster lunifer (Lepidoptera, Notodontidae),” BMC Genomics, vol. 9, p. 331, 2008. View at: Publisher Site | Google Scholar
  37. X. C. Wang, X. Y. Sun, Q. Q. Sun et al., “The complete mitochondrial genome of the laced fritillary Argyreus hyperbius (Lepidoptera: Nymphalidae),” Zoology Research, vol. 32, no. 5, pp. 465–475, 2011. View at: Google Scholar
  38. H. N. Chai, Y. Z. Du, and B. P. Zhai, “Characterization of the complete mitochondrial genomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae),” International Journal of Biological Sciences, vol. 8, no. 4, pp. 561–579, 2012. View at: Google Scholar
  39. Z. H. Mao, J. S. Hao, G. P. Zhu, J. Hu, M. M. Si, and C. D. Zhu, “Sequencing and analysis of the complete mitochondrial genome of Pieris rapae Linnaeus (Lepidoptera: Pieridae),” Acta Entomologica Sinica, vol. 53, no. 11, pp. 1295–1304, 2010. View at: Google Scholar
  40. C. R. Fauron and D. R. Wolstenholme, “Extensive diversity among Drosophila species with respect to nucleotide sequences within the adenine + thymine-rich region of mitochondrial DNA molecules,” Nucleic Acids Research, vol. 8, no. 11, pp. 2439–2452, 1980. View at: Publisher Site | Google Scholar
  41. D. O. Clary and D. R. Wolstenholme, “Drosophila mitochondrial DNA: conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA,” Journal of Molecular Evolution, vol. 25, no. 2, pp. 116–125, 1987. View at: Publisher Site | Google Scholar
  42. K. Yukuhiro, H. Sezutsu, M. Itoh, K. Shimizu, and Y. Banno, “Significant levels of sequence divergence and gene rearrangements have occurred between the mitochondrial genomes of the wild mulberry silkmoth, Bombyx mandarina, and its close relative, the domesticated silkmoth, Bombyx mori,” Molecular Biology and Evolution, vol. 19, no. 8, pp. 1385–1389, 2002. View at: Google Scholar
  43. E. S. Lee, K. S. Shin, M. S. Kim, H. Park, S. Cho, and C. B. Kim, “The mitochondrial genome of the smaller tea tortrix Adoxophyes honmai (Lepidoptera: Tortricidae),” Gene, vol. 373, no. 1-2, pp. 52–57, 2006. View at: Publisher Site | Google Scholar
  44. S. R. Kim, M. I. Kim, M. Y. Hong et al., “The complete mitogenome sequence of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae),” Molecular Biology Reports, vol. 36, no. 7, pp. 1871–1880, 2009. View at: Publisher Site | Google Scholar
  45. L. Yang, Z. J. Wei, G. Y. Hong, S. T. Jiang, and L. P. Wen, “The complete nucleotide sequence of the mitochondrial genome of Phthonandria atrilineata (Lepidoptera: Geometridae),” Molecular Biology Reports, vol. 36, no. 6, pp. 1441–1449, 2009. View at: Publisher Site | Google Scholar
  46. S. L. Cameron and M. F. Whiting, “The complete mitochondrial genome of the tobacco hornworm, Manduca sexta, (Insecta: Lepidoptera: Sphingidae), and an examination of mitochondrial gene variability within butterflies and moths,” Gene, vol. 408, no. 1-2, pp. 112–123, 2008. View at: Publisher Site | Google Scholar
  47. M. Y. Hong, E. M. Lee, Y. H. Jo et al., “Complete nucleotide sequence and organization of the mitogenome of the silk moth Caligula boisduvalii (Lepidoptera: Saturniidae) and comparison with other lepidopteran insects,” Gene, vol. 413, no. 1-2, pp. 49–57, 2008. View at: Publisher Site | Google Scholar
  48. Y. Liu, Y. Li, M. Pan et al., “The complete mitochondrial genome of the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae),” Acta Biochimica et Biophysica Sinica, vol. 40, no. 8, pp. 693–703, 2008. View at: Publisher Site | Google Scholar
  49. L. W. Wu, D. C. Lees, S. H. Yen, C. C. Lu, and Y. F. Hsu, “complete mitochondrial genome of the near-threatened swallowtail, Agehana maraho (Lepidoptera: Papilionidae): evaluating sequence variability and suitable markers for conservation genetic studies,” Entomological News, vol. 121, no. 3, pp. 267–280, 2010. View at: Publisher Site | Google Scholar
  50. M. Chen, L. L. Tian, Q. H. Shi, T. W. Cao, and J. S. Hao, “Complete mitogenome of the lesser purple emperor Apatura ilia (Lepidoptera: Nymphalidae: Apaturinae) and comparison with other nymphalid butterflies,” Zoology Research, vol. 33, no. 2, pp. 191–201, 2012. View at: Google Scholar
  51. S. Saito, K. Tamuea, and T. Aotsuka, “Replicatiion origin of mitochondrial DNA in insects,” Genetics, vol. 171, pp. 433–448, 2005. View at: Google Scholar
  52. N. Wahlberg, E. Weingartner, and S. Nylin, “Towards a better understanding of the higher systematics of Nymphalidae (Lepidoptera: Papilionoidea),” Molecular Phylogenetics and Evolution, vol. 28, no. 3, pp. 473–484, 2003. View at: Publisher Site | Google Scholar
  53. A. V. L. Freitas and K. S. Brown, “Phylogeny of the Nymphalidae (Lepidoptera),” Systematic Biology, vol. 53, no. 3, pp. 363–383, 2004. View at: Publisher Site | Google Scholar
  54. J. S. Hao, C. Y. Su, G. P. Zhu, N. Chen, D. X. Wu, and X. P. Zhang, “The molecular morphologies of mitochondrial 16S rRNA of the main butterfly lineages and their phylogenetic significances,” Journal of Genetics and Molecular Biology, vol. 18, no. 2, pp. 111–113, 2007. View at: Google Scholar
  55. N. P. Kristensen, “Remarks on the family-level phylogeny of butterflies (Insecta, Lepidoptera, Rhopalocera),” Zoological Systematics and Evolutionary Research, vol. 14, no. 1, pp. 25–33, 1976. View at: Google Scholar
  56. R. De Jong, R. I. Vane-Wright, and P. R. Ackery, “The higher classification of butterflies (Lepidoptera): problems and prospects,” Insect Systematics and Evolution, vol. 27, no. 1, pp. 65–101, 1996. View at: Google Scholar
  57. R. I. Vane-Wright, “Evidence and identity in butterfly systematics,” in Butterflies: Ecology and Evolution Taking Flight, C. L. Boggs, W. B. Watt, and P. R. Ehrlich, Eds., pp. 477–513, University of Chicago Press, Chicago, 2003. View at: Google Scholar
  58. A. Pagenstecher, “Lepidoptera rhopalocera: family libytheidae,” in Fascicle, P. Wytsman, Ed., vol. 5, pp. 1–4, Genera Insectorum, Bruxelles, Belgium, 1902. View at: Google Scholar
  59. A. Pagenstecher, “Libytheidae,” in Lepidopterorum Catalogus, C. Aurivillius and H. Wagner, Eds., R. Friedländer und sohn, Berlin, Germany, 1911. View at: Google Scholar
  60. A. V. Z. Brower, “Phylogenetic relationships among the Nymphalidae (Lepidoptera) inferred from partial sequences of the wingless gene,” Proceedings of the Royal Society B, vol. 267, no. 1449, pp. 1201–1211, 2000. View at: Google Scholar
  61. P. R. Ehrlich, “The comparative morphology, phylogeny, and higher classification of the butterflies (Lepidoptera: Papilionoidea),” University of Kansas Science Bulletin, vol. 39, pp. 305–370, 1958. View at: Google Scholar
  62. A. Y. Kawahara, “Phylogeny of snout butterflies (Lepidoptera: Nymphalidae: Libytheinae): combining evidence from the morphology of extant, fossil, and recently extinct taxa,” Cladistics, vol. 25, no. 3, pp. 263–278, 2009. View at: Publisher Site | Google Scholar
  63. X. Feng, D. F. Liu, N. X. Wang, C. D. Zhu, and G. F. Jiang, “The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses,” Molecular Biology Reports, vol. 37, no. 8, pp. 3877–3888, 2010. View at: Publisher Site | Google Scholar
  64. F. Qin, G. F. Jiang, and S. Y. Zhou, “Complete mitochondrial genome of the Teinopalpus aureus guangxiensis (Lepidoptera: Papilionidae) and related phylogenetic analyses,” Mitochondrial DNA, vol. 23, no. 2, pp. 123–125, 2012. View at: Publisher Site | Google Scholar
  65. M. J. Kim, R. A. Kang, H. C. Jeong, K. G. Kim, and I. Kim, “Reconstructing intraordinal relationships in Lepidoptera using mitochondrial genome data with the description of two newly sequenced lycaenids, Spindasis takanonis and Protantigius superans (Lepidoptera: Lycaenidae),” Molecular Phylogenetics and Evolution, vol. 61, no. 2, pp. 436–445, 2011. View at: Publisher Site | Google Scholar
  66. X. M. Qin, Q. X. Guan, D. L. Zeng, F. Qin, and H. M. Li, “Complete mitochondrial genome of Kallima inachus (Lepidoptera: Nymphalidae: Nymphalinae): comparison of K. inachus and Argynnis hyperbius,” Mitochondrial DNA, vol. 23, no. 4, pp. 318–320, 2012. View at: Publisher Site | Google Scholar
  67. J. Xia, J. Hu, G. P. Zhu, C. D. Zhu, and J. S. Hao, “Complete mitochondrial DNA sequence of the Calinaga davidis,” Acta Entomologica Sinica, vol. 54, no. 5, pp. 555–565, 2011. View at: Google Scholar
  68. M. J. Kim, X. Wan, K. G. Kim, J. S. Hwang, and I. Kim, “Complete nucleotide sequence and organization of the mitogenome of endangered Eumenis autonoe (Lepidoptera: Nymphalidae),” African Journal of Biotechnology, vol. 9, no. 5, pp. 735–754, 2010. View at: Google Scholar
  69. Y. J. Zhu, R. Fang, G. L. Zhou, J. Ye, and J. P. Yi, “The complete sequence determination and analysis of Lymantria dispar genome,” Plant Quarantine, vol. 24, no. 4, pp. 6–11, 2010. View at: Google Scholar

Copyright © 2013 Jiasheng Hao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1635 Views | 737 Downloads | 27 Citations
 PDF  Download Citation  Citation
 Download other formatsMore
 Order printed copiesOrder

We are committed to sharing findings related to COVID-19 as quickly and safely as possible. Any author submitting a COVID-19 paper should notify us at to ensure their research is fast-tracked and made available on a preprint server as soon as possible. We will be providing unlimited waivers of publication charges for accepted articles related to COVID-19.