# Complete Mitogenomes ofEuploea mulciber (Nymphalidae: Danainae) and Libythea celtis (Nymphalidae: Libytheinae) and Their Phylogenetic Implications

Authors: Jiasheng Hao; Minǵe Sun; Qinghui Shi; Xiaoyan Sun; Lili Shao; Qun Yang
Journal: ISRN Genomics
 (2013)

---

## Abstract

The complete mitochondrial genome sequences of the two butterfly speciesEuploea 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.


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## Body

## 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 [6–8]. 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 [12–19]. 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 ofEuploea 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 (Table1), 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.Table 1
The available rhopaloceran mitogenomes (two new and 22 obtained from GenBank) and their characteristics.
Whole genome
PCGb
lrRNA
srRNA
A + T-rich region
Family/subfamily
Species
Size
A + T
No. codonsa
A + T
Size
A + T
Size
A + T
Size
A + T
Gen of Bank accession no.
References
(bp)
(%)
(%)
(bp)
(%)
(bp)
(%)
(bp)
(%)
Papilionidae/Papilioninae
Troides aeacus
15,263
80.2
3745
79.1
1234
83.3
784
85.0
419
89.8
EU625344
Unpublished
Papilio xuthus
13,964
80.0
3583
78.9
1327
83.6
598
84.5
—
—
EF176224
[63]
Agehana maraho
16,094
80.5
3718
79.2
1333
83.7
779
85.5
1270
95.0
FJ810212
[49]
Teinopalpus aureus
15,242
79.8
3720
78.3
1320
82.4
781
85.6
395
93.1
HM563681
[64]
Parnassius bremeri
15,389
81.3
3723
80.2
1344
83.9
773
85.1
504
93.6
FJ871125
[13]
Papilionidae/Parnassiinae
Sericinus montela
15,242
80.8
3691
80.2
1338
83.6
759
84.6
408
94.1
HQ259122
[18]
Luehdorfia chinensis
13,860
80.5
3551
79.6
1342
84.0
460
81.5
—
—
EU622524
Unpublished
Hesperiidae/Pyrginae
Ctenoptilum vasava
15,468
80.5
3698
78.9
1343
84.1
774
86.4
429
88.1
JF713818
[19]
Pieridae/Pierinae
Artogeia melete
15,140
79.8
3715
78.4
1319
83.4
777
85.5
351
89.2
NC_010568
[14]
Pieris rapae
15,157
79.7
3722
78.3
1320
84.0
764
85.0
393
91.6
HM156697
[39]
Lycaenidae/Theclinae
Coreana raphaelis
15,314
82.7
3708
81.5
1330
85.3
777
85.8
375
94.1
DQ102703
[12]
Protantigius superans
15,248
81.7
3712
80.3
1331
85.1
739
85.6
361
93.6
HQ184265
[65]
Lycaenidae/Aphnaeinae
Spindasis takanonis
15,349
82.4
3719
81.0
1333
85.6
777
84.7
371
94.6
HQ184266
[65]
Apatura ilia
15,242
80.5
3711
78.9
1333
84.0
776
84.9
403
92.5
JF439725
[50]
Nymphalidae/Apaturinae
Sasakia charonda
15,244
79.9
3695
78.2
1323
84.4
775
85.0
380
91.8
NC_014224
Unpublished
Sasakia
c
h
a
r
o
n
d
a
*
15,222
79.9
3586
78.3
1311
84.2
775
85.0
380
91.8
NC_014223
Unpublished
Nymphalidae/Nymphalinae
Kallima inachus
15,183
80.3
3721
79.2
1335
82.7
774
85.1
376
92.0
JN857943
[66]
Nymphalidae/Heliconiinae
Argyreus hyperbius
15,156
80.8
3718
79.5
1330
84.5
778
85.2
349
95.4
JF439070
[37]
Acraea issoria
15,245
79.7
3717
78.0
1331
83.9
788
83.7
430
96.0
GQ376195
[15]
Nymphalidae/Limenitidinae
Parathyma sulpitia
15,268
81.9
3729
80.6
1319
84.7
779
85.7
349
94.6
JQ347260
[17]
Nymphalidae/Calinaginae
Calinaga davidis
15,267
80.4
3737
78.9
1337
83.8
773
85.9
389
92.0
NC_015480
[67]
Nymphalidae/Danainae
Eumenis autonoe
15,489
79.1
3728
76.8
1335
83.7
775
85.3
678
94.5
GQ868707
[68]
Nymphalidae/Libytheinae
Libythea celtis
15,164
81.2
3722
80.0
1335
84.7
775
85.4
328
96.3
HQ378508
This study
Nymphalidae/Danainae
Euploea mulciber
15,166
81.5
3705
80.2
1314
84.6
776
85.3
399
93.5
HQ37507
This study
Lymantriidae
Lymantria dispar
15,569
79.9
3742
77.8
1351
84.2
799
85.2
435
96.1
NC_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.


## 2.1. Specimen Collection

Adult individuals ofEuploea 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 (Table1), 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.Table 1
The available rhopaloceran mitogenomes (two new and 22 obtained from GenBank) and their characteristics.
Whole genome
PCGb
lrRNA
srRNA
A + T-rich region
Family/subfamily
Species
Size
A + T
No. codonsa
A + T
Size
A + T
Size
A + T
Size
A + T
Gen of Bank accession no.
References
(bp)
(%)
(%)
(bp)
(%)
(bp)
(%)
(bp)
(%)
Papilionidae/Papilioninae
Troides aeacus
15,263
80.2
3745
79.1
1234
83.3
784
85.0
419
89.8
EU625344
Unpublished
Papilio xuthus
13,964
80.0
3583
78.9
1327
83.6
598
84.5
—
—
EF176224
[63]
Agehana maraho
16,094
80.5
3718
79.2
1333
83.7
779
85.5
1270
95.0
FJ810212
[49]
Teinopalpus aureus
15,242
79.8
3720
78.3
1320
82.4
781
85.6
395
93.1
HM563681
[64]
Parnassius bremeri
15,389
81.3
3723
80.2
1344
83.9
773
85.1
504
93.6
FJ871125
[13]
Papilionidae/Parnassiinae
Sericinus montela
15,242
80.8
3691
80.2
1338
83.6
759
84.6
408
94.1
HQ259122
[18]
Luehdorfia chinensis
13,860
80.5
3551
79.6
1342
84.0
460
81.5
—
—
EU622524
Unpublished
Hesperiidae/Pyrginae
Ctenoptilum vasava
15,468
80.5
3698
78.9
1343
84.1
774
86.4
429
88.1
JF713818
[19]
Pieridae/Pierinae
Artogeia melete
15,140
79.8
3715
78.4
1319
83.4
777
85.5
351
89.2
NC_010568
[14]
Pieris rapae
15,157
79.7
3722
78.3
1320
84.0
764
85.0
393
91.6
HM156697
[39]
Lycaenidae/Theclinae
Coreana raphaelis
15,314
82.7
3708
81.5
1330
85.3
777
85.8
375
94.1
DQ102703
[12]
Protantigius superans
15,248
81.7
3712
80.3
1331
85.1
739
85.6
361
93.6
HQ184265
[65]
Lycaenidae/Aphnaeinae
Spindasis takanonis
15,349
82.4
3719
81.0
1333
85.6
777
84.7
371
94.6
HQ184266
[65]
Apatura ilia
15,242
80.5
3711
78.9
1333
84.0
776
84.9
403
92.5
JF439725
[50]
Nymphalidae/Apaturinae
Sasakia charonda
15,244
79.9
3695
78.2
1323
84.4
775
85.0
380
91.8
NC_014224
Unpublished
Sasakia
c
h
a
r
o
n
d
a
*
15,222
79.9
3586
78.3
1311
84.2
775
85.0
380
91.8
NC_014223
Unpublished
Nymphalidae/Nymphalinae
Kallima inachus
15,183
80.3
3721
79.2
1335
82.7
774
85.1
376
92.0
JN857943
[66]
Nymphalidae/Heliconiinae
Argyreus hyperbius
15,156
80.8
3718
79.5
1330
84.5
778
85.2
349
95.4
JF439070
[37]
Acraea issoria
15,245
79.7
3717
78.0
1331
83.9
788
83.7
430
96.0
GQ376195
[15]
Nymphalidae/Limenitidinae
Parathyma sulpitia
15,268
81.9
3729
80.6
1319
84.7
779
85.7
349
94.6
JQ347260
[17]
Nymphalidae/Calinaginae
Calinaga davidis
15,267
80.4
3737
78.9
1337
83.8
773
85.9
389
92.0
NC_015480
[67]
Nymphalidae/Danainae
Eumenis autonoe
15,489
79.1
3728
76.8
1335
83.7
775
85.3
678
94.5
GQ868707
[68]
Nymphalidae/Libytheinae
Libythea celtis
15,164
81.2
3722
80.0
1335
84.7
775
85.4
328
96.3
HQ378508
This study
Nymphalidae/Danainae
Euploea mulciber
15,166
81.5
3705
80.2
1314
84.6
776
85.3
399
93.5
HQ37507
This study
Lymantriidae
Lymantria dispar
15,569
79.9
3742
77.8
1351
84.2
799
85.2
435
96.1
NC_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 ofEuploea 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 tRNAGln  and ND2 (46 bp), between ND5 and tRNAHis (15 bp), and between tRNASer (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 tRNAGln and ND2 (52 bp) and between tRNASer (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).Table 2
Summary of the mitogenomes ofEuploea mulciberandLibythea celtis.
Gene
Direction
Nucleotide no.
Size (bp)
Intergenic nucleotides
Anticodon
Start codon
Stop codon
Species
1*
2*
1
2
1
2
1
2
1
2
1
2
1
2
tRNAMet
F
F
1–68
1–68
68
68
32–34 CAT
32–34 CAT
tRNAIle(AUR)
F
F
78–140
67–131
63
65
9
−2
106–108 GAT
95–97 GAT
tRNAGln
R
R
138–206
129–197
69
69
−3
−3
174–176 TTG
165–167 TTG
ND2
F
F
253–1251
250–1263
999
1014
46
52
ATT
ATC
TAA
TAA
tRNATrp
F
F
1250–1317
1265–1331
68
67
−2
1
1281–1283 TCA
1296–1298 TCA
tRNACys
R
R
1310–1372
1324–1386
63
63
−8
−8
1340–1342 GCA
1355–1357 GCA
tRNATyr
R
R
1374–1439
1387–1451
66
65
1
0
1406–1408 GTA
1418–1420 GTA
COI
F
F
1442–2972
1458–2988
1531
1531
2
6
CGA
CGA
T-tRNALeu(UUR)
T-tRNALeu(UUR)
tRNALeu(UUR)
F
F
2973–3039
2989–3055
67
67
0
0
3003–3005 TAA
3019–3021 TAA
COII
F
F
3040–3712
3056–3737
673
682
0
0
ATG
ATG
T-tRNALys
T-tRNALys
tRNALys
F
F
3713–3783
3738–3808
71
71
0
0
3743–3745 CTT
3768–3770 CTT
tRNAAsp
F
F
3783–3848
3808–3873
66
66
−1
−1
3813–3815 GTC
3838–3840 GTC
ATP8
F
F
3849–4010
3874–4041
162
168
0
0
ATT
ATT
TAA
TAA
ATP6
F
F
4004–4681
4035–4712
678
678
−7
−7
ATG
ATG
TAA
TAA
COIII
F
F
4681–5469
4712–5500
789
789
−1
−1
ATG
ATG
TAA
TAA
tRNAGly
F
F
5476–5541
5503–5569
66
67
6
2
5506–5508 TCC
5533–5535 TCC
ND3
F
F
5542–5895
5570–5923
354
354
0
0
ATT
ATT
TAG
TAA
tRNAAla
F
F
5894–5959
5928–5996
66
69
−2
4
5925–5927 TGC
5961–5963 TGC
tRNAArg
F
F
5959–6024
5997–6059
66
63
−1
0
5987–5989 TCG
6024–6026 TCG
tRNAAsn
F
F
6026–6091
6065–6130
66
66
1
5
6057–6059 GTT
6096–6098 GTT
tRNASer(AGN)
F
F
6090–6149
6129–6190
60
62
−2
−2
6110–6112 TCT
6150–6152 GCT
tRNAGlu
F
F
6158–6223
6192–6259
66
68
8
1
6188–6190 TTC
6224–6226 TTC
tRNAPhe
R
R
6222–6286
6258–6325
65
68
−2
−2
6254–6256 GAA
6290–6292 GAA
ND5
R
R
6287–8003
6326–8057
1717
1732
0
0
ATT
ATT
T-tRNAPhe
T-tRNAPhe
tRNAHis
R
R
8019–8083
8058–8122
65
65
15
0
8051–8053 GTG
8090–8092 GTG
ND4
R
R
8084–9422
8123–9461
1339
1339
0
0
ATG
ATG
T-tRNAHis
T-tRNAHis
ND4L
R
R
9422–9709
9464–9754
288
291
−1
2
ATG
ATG
TAA
TAG
tRNAThr
F
F
9712–9775
9757–9821
64
65
2
2
9742–9744 TGT
9788–9790 TGT
tRNAPro
R
R
9776–9839
9822–9886
64
65
0
0
9808–9810 TGG
9854–9856 TGG
ND6
F
F
9842–10366
9889–10419
525
531
2
2
ATA
ATT
TAA
TAA
CytB
F
F
10371–11,519
10419–11,567
1149
1149
4
−1
ATG
ATG
TAA
TAA
tRNASer(UCN)
F
F
11,518–11,582
11,570–11,636
65
67
−2
2
11547–11549 TGA
11600–11602 TGA
ND1
R
R
11,603–12,544
11,654–12,592
942
939
20
17
ATG
ATG
TAA
TAA
tRNALeu(CUN)
R
R
12,546–12,612
12,594–12,660
67
67
1
1
12581–12583 TAG
12629–12631 TAG
lrRNA
R
R
12,613–13,926
12,661–13,995
1314
1335
0
0
tRNAVal
R
R
13,927–13,991
13,996–14,061
65
66
0
0
13961–13963 TAC
14029–14031 TAC
srRNA
R
R
13,992–14,767
14,062–14,836
776
775
0
0
D-loop
F
F
14,768–15,166
14,837–15,164
399
328
0
0
1* indicates the species ofEuploea mulciberand  2* indicates the species of Libythea celtis.Figure 1
Map of the mitogenomes ofEuploea mulciber and Libythea celtis. The abbreviations for the genes are as follows: ATP6, ATP8, ATP: synthase subunits 6 and 8 genes; CytB: cytochrome oxidase b gene; COI–COIII: cytochrome oxidase c subunit 1–3 genes; ND1–6, ND4L, NADH: dehydrogenase subunits 1–6 and 4L. Gene names are the standard abbreviations used in this paper; tRNA genes are denoted as one-letter symbol according to the IUPAC-IUB single letter amino acid codes. Gene names that are not underlined indicate the direction of transcription clockwise, while the underlining indicates counterclockwise.


### 3.2. Protein-Coding Genes

The total codon numbers of the 13 PCGs forEuploea 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, 36–38].Table 3
Nucleotide composition at each codon position of the concatenated 13 PCGs in the Papilionoidea mitogenome.
Species
1st  codon  position
2nd  codon  position
3rd  codon  position
Overall
A
T
C
G
A
T
C
G
A
T
C
G
A
T
C
G
Papilio xuthus
36.9
37.2
10.5
15.4
21.9
48.9
16.2
13.0
40.6
48.9
6.5
3.9
33.1
45.0
11.1
10.8
Agehana maraho
36.9
37.2
10.5
15.4
21.9
48.9
16.2
13.0
40.6
48.9
6.5
3.9
33.1
45.0
11.1
10.8
Troides aeacus
36.6
37.0
10.3
16.1
22.5
48.4
15.9
13.2
40.5
52.2
4.9
2.4
33.2
45.8
10.4
10.6
Sericinus montela
37.5
37.8
9.6
15.1
22.0
48.9
16.2
13.0
43.1
50.1
4.3
2.5
34.2
45.6
10.0
10.2
Teinopalpus aureus
37.0
37.0
10.4
15.6
21.9
48.2
16.6
13.3
40.6
50.2
5.5
3.7
33.1
45.1
10.8
10.9
Luehdorfia chinensis
36.6
37.4
10.1
15.9
22.2
48.5
16.2
13.1
42.7
51.3
3.7
2.3
33.8
45.7
10.0
10.4
Parnassius bremeri
37.0
37.9
9.4
15.7
22.2
48.4
16.4
13.0
44.1
51.0
3.3
1.7
34.4
45.8
9.7
10.2
Coreana raphaelis
38.3
38.1
8.8
14.9
22.0
49.0
15.8
13.2
45.8
51.2
2.0
1.0
35.4
46.1
8.8
9.7
Spindasis takanonis
38.6
38.2
9.2
14.1
22.5
49.4
15.2
12.9
43.6
51.0
2.8
2.7
34.9
46.2
9.1
9.9
Protantigius superans
37.7
37.7
9.2
15.3
22.0
48.8
15.9
13.2
44.0
50.6
3.2
2.2
34.6
45.7
9.4
10.2
Pieris rapae
36.0
37.6
9.9
16.5
22.1
48.0
16.6
13.4
42.7
48.2
5.3
3.8
33.6
44.6
10.6
11.2
Artogeia melete
36.7
36.8
10.3
16.2
21.9
47.9
16.8
13.4
42.3
49.8
4.6
3.4
33.6
44.8
10.6
11.0
Apatura ilia
37.2
37.6
9.6
15.6
22.0
48.8
15.9
13.2
41.7
49.7
5.3
3.3
33.6
45.4
10.3
10.7
Sasakia charonda
36.7
37.6
9.8
15.9
22.3
48.3
16.1
13.3
40.9
48.5
6.1
4.4
33.3
44.8
10.7
11.2
Sasakia
c
h
a
r
o
n
d
a
*
36.8
37.7
9.7
15.8
22.2
48.5
16.1
13.2
40.6
48.8
6.0
4.6
33.2
45.0
10.6
11.2
Kallima inachus
36.4
37.2
10.1
16.3
21.8
48.5
16.4
13.3
42.5
51.2
3.5
2.7
33.6
45.6
10.0
10.8
Argyreus hyperbius
37.4
37.1
9.9
15.6
22.6
48.2
6.1
13.1
41.5
51.5
4.6
2.4
33.8
45.6
10.2
10.4
Acraea issoria
36.6
36.7
10.7
15.9
23.0
47.8
16.2
13.1
39.7
50.1
6.4
3.7
33.1
44.9
11.1
10.9
Parathyma sulpitia
36.9
37.9
9.8
15.4
22.3
48.2
16.4
13.1
42.0
54.7
2.1
1.2
33.7
46.9
9.4
9.9
Calinaga davidis
36.3
37.7
10.4
15.7
22.2
48.4
16.4
13.1
40.7
51.5
5.1
2.7
33.0
45.8
10.6
10.5
Eumenis autonoe
35.9
36.3
11.3
16.4
21.5
48.4
16.4
13.7
39.7
48.8
7.1
4.4
32.3
44.5
11.6
11.5
Euploea mulciber
37.4
37.4
9.5
15.7
21.9
48.5
16.5
13.1
44.1
51.3
3.0
1.6
34.5
45.7
9.7
10.1
Libythea celtis
36.6
37.6
9.6
16.2
22.0
48.2
16.5
13.3
44.1
51.4
2.6
1.9
34.2
45.7
9.6
10.4
Ctenoptilum vasava
36.1
38.1
10.0
15.7
22.3
48.0
16.7
13.0
41.0
51.2
4.9
2.9
33.1
45.8
10.5
10.5
Start codons and stop codons were excluded in the count.*Sasakia charonda kuriyamaensis.Table 4
Codon usage of the protein-coding genes of theLibythea celtis and Euploea mulcibermitogenomes.
Codon
N (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)
N: frequency of codon used; RSCU: relative synonymous codon usage; *stop codon.
Start codons and stop codons were excluded in total codon counts.Table 5
Nucleotide composition and skewness in the rhopaloceran mitogenomes.
Major strand PCGs
Minor strand PCGs
Whole PCGs
Whole genome
Species
A + T
AT skew
GC skew
A + T
AT skew
GC skew
A + T
AT skew
GC skew
A + T
AT skew
GC skew
%
%
%
%
Papilio xuthus
77.1
-
0.114
-
0.206
79.8
-
0.210
0.334
78.1
-
0.152
-
0.014
80.0
-
0.011
-
0.216
Agehana maraho
77.1
-
0.114
-
0.206
79.8
-
0.210
0.334
78.1
-
0.152
-
0.014
80.6
0.006
-
0.262
Teinopalpus aureus
77.2
-
0.125
-
0.172
80.0
-
0.196
0.322
78.3
-
0.153
0.002
79.8
-
0.005
-
0.238
Troides aeacus
77.7
-
0.166
-
0.168
81.1
-
0.150
0.332
79.1
-
0.160
0.010
80.2
-
0.040
-
0.230
Parnassius bremeri
78.9
-
0.126
-
0.112
82.2
-
0.166
0.281
80.2
-
0.142
0.024
81.3
−0.011
−0.194
Sericinus montela
78.5
-
0.124
-
0.146
81.8
-
0.172
0.301
79.8
-
0.143
0.009
81.0
-
0.008
-
0.223
Luehdorfia chinensis
78.0
-
0.129
-
0.134
81.9
-
0.179
0.296
79.6
-
0.150
0.019
80.5
-
0.005
-
0.205
Ctenoptilum vasava
77.2
-
0.151
-
0.145
81.6
-
0.174
0.285
78.9
-
0.160
0.000
80.5
-
0.029
-
0.205
Pieris rapae
77.2
-
0.095
-
0.150
79.8
-
0.212
0.358
78.2
-
0.141
0.030
79.7
0.014
-
0.232
Artogeia melete
77.7
-
0.098
-
0.152
79.7
-
0.213
0.324
78.4
-
0.143
0.020
79.8
0.012
−0.222
Coreana raphaelis
80.3
-
0.143
-
0.074
83.4
-
0.115
0.270
81.5
-
0.132
0.044
82.7
-
0.047
-
0.158
Protantigius superans
79.3
-
0.137
-
0.087
81.9
-
0.140
0.276
80.3
-
0.138
0.041
81.7
-
0.036
-
0.174
Spindasis takanonis
80.1
-
0.104
-
0.117
82.7
-
0.193
0.338
81.1
-
0.139
0.044
82.4
0.004
-
0.214
Apatura ilia
77.9
-
0.124
-
0.140
80.7
-
0.187
0.314
79.0
-
0.149
0.021
80.4
-
0.011
-
0.207
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
c
h
a
r
o
n
d
a
*
77.2
-
0.121
-
0.142
79.7
-
0.196
0.324
78.2
-
0.151
0.028
79.9
-
0.007
-
0.218
Kallima inachus
78.1
-
0.136
-
0.125
81.1
-
0.177
0.336
79.2
-
0.152
0.036
80.3
-
0.013
-
0.212
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
-
0.124
-
0.094
81.8
-
0.174
0.297
79.9
-
0.144
0.040
81.2
-
0.017
-
0.181
Euploea mulciber
79.0
-
0.142
-
0.124
82.1
-
0.140
0.307
80.2
-
0.140
0.020
81.5
-
0.038
-
0.211
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 inEuploea 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 Figure2, 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 tRNATyr. 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 tRNATyr. 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].Figure 2
Alignment of the initiation context of the COI genes of 25 lepidopteran insect species, including that ofLibythea celtis and Euploea mulciber.The first several codons for COI and their amino acids are shown on the right-hand side of the figure. Wave-lined nucleotides indicate the adjacent partial sequence of tRNATyr. Arrows indicate the transcriptional direction. Boxed nucleotides indicate the currently proposed translation initiators for the COI gene of lepidopteran insects. The start codons for Libythea celtisand Euploea mulciberare designated as CGA. *Sasakia charonda kuriyamaensis.Nine of the PCGs inEuploea 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 theEuploea 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 tRNALeu (CUN) and tRNAVal genes [12, 13, 15, 16, 19, 37, 39–45]. 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 tRNAVal 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, bothEuploea 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 tRNASer (AGN), whose dihydrouridine (DHU) arm forms a simple loop (maps of secondary structure are not shown here). The case of tRNASer (AGN) with the simple loop in the DHU arm has also been detected in most of insect species including the lepidopterans determined till now [12–19, 36, 37, 39, 42, 45–48].


### 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. TheEuploea mulciber and Libythea celtis A + T-rich regions located between srRNA and tRNAMet were 399 and 328 bp in length, respectively (see Figure 1 and Table 2). Though the A + T-rich region ofLibythea 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 tRNAMet.Alignment of the initiation sites for the A + T-rich regions of 22 completely sequenced rhopaloceran mitogenomes is shown in Figure3, 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 inAgehana 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].Figure 3
Alignment of the initiation site for the A + T-rich regions of 23 completely sequenced lepidopteran mitogenomes. The shaded nucleotides indicate the poly-T stretch, and the boxed nucleotides indicate the conserved motif, ATAGA. *Sasakia charonda kuriyamaensis.A microsatellite-like elements (AT)3(TA)6 and (TA)12 preceded by the ATTTA motif are presented in the middle of theEuploea mulciber and Libythea celtis A + T-rich region, respectively. Similar cases are commonly found in all other sequenced lepidopteran species [12–15, 17, 19, 36–39, 42–44, 46–48]. Additionally, a 12 bp poly A interrupted by T and a 7 bp poly A interrupted by G presented immediately the upstream of the tRNAMet. 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 Figure4 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 totheCalinaga davidis(Calinaginae).NJ tree (a) and BI tree (b) of the main lineages of butterflies based on nucleotide sequences of the 13 protein-coding genes; numbers at the nodes: bootstrap values in (a) and posterior probabilities in (b).
(a)
(b)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, 55–57]. 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, 60–62]. 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.


## 3.1. Genome Organization and Structure

The mitogenomes ofEuploea 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 tRNAGln  and ND2 (46 bp), between ND5 and tRNAHis (15 bp), and between tRNASer (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 tRNAGln and ND2 (52 bp) and between tRNASer (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).Table 2
Summary of the mitogenomes ofEuploea mulciberandLibythea celtis.
Gene
Direction
Nucleotide no.
Size (bp)
Intergenic nucleotides
Anticodon
Start codon
Stop codon
Species
1*
2*
1
2
1
2
1
2
1
2
1
2
1
2
tRNAMet
F
F
1–68
1–68
68
68
32–34 CAT
32–34 CAT
tRNAIle(AUR)
F
F
78–140
67–131
63
65
9
−2
106–108 GAT
95–97 GAT
tRNAGln
R
R
138–206
129–197
69
69
−3
−3
174–176 TTG
165–167 TTG
ND2
F
F
253–1251
250–1263
999
1014
46
52
ATT
ATC
TAA
TAA
tRNATrp
F
F
1250–1317
1265–1331
68
67
−2
1
1281–1283 TCA
1296–1298 TCA
tRNACys
R
R
1310–1372
1324–1386
63
63
−8
−8
1340–1342 GCA
1355–1357 GCA
tRNATyr
R
R
1374–1439
1387–1451
66
65
1
0
1406–1408 GTA
1418–1420 GTA
COI
F
F
1442–2972
1458–2988
1531
1531
2
6
CGA
CGA
T-tRNALeu(UUR)
T-tRNALeu(UUR)
tRNALeu(UUR)
F
F
2973–3039
2989–3055
67
67
0
0
3003–3005 TAA
3019–3021 TAA
COII
F
F
3040–3712
3056–3737
673
682
0
0
ATG
ATG
T-tRNALys
T-tRNALys
tRNALys
F
F
3713–3783
3738–3808
71
71
0
0
3743–3745 CTT
3768–3770 CTT
tRNAAsp
F
F
3783–3848
3808–3873
66
66
−1
−1
3813–3815 GTC
3838–3840 GTC
ATP8
F
F
3849–4010
3874–4041
162
168
0
0
ATT
ATT
TAA
TAA
ATP6
F
F
4004–4681
4035–4712
678
678
−7
−7
ATG
ATG
TAA
TAA
COIII
F
F
4681–5469
4712–5500
789
789
−1
−1
ATG
ATG
TAA
TAA
tRNAGly
F
F
5476–5541
5503–5569
66
67
6
2
5506–5508 TCC
5533–5535 TCC
ND3
F
F
5542–5895
5570–5923
354
354
0
0
ATT
ATT
TAG
TAA
tRNAAla
F
F
5894–5959
5928–5996
66
69
−2
4
5925–5927 TGC
5961–5963 TGC
tRNAArg
F
F
5959–6024
5997–6059
66
63
−1
0
5987–5989 TCG
6024–6026 TCG
tRNAAsn
F
F
6026–6091
6065–6130
66
66
1
5
6057–6059 GTT
6096–6098 GTT
tRNASer(AGN)
F
F
6090–6149
6129–6190
60
62
−2
−2
6110–6112 TCT
6150–6152 GCT
tRNAGlu
F
F
6158–6223
6192–6259
66
68
8
1
6188–6190 TTC
6224–6226 TTC
tRNAPhe
R
R
6222–6286
6258–6325
65
68
−2
−2
6254–6256 GAA
6290–6292 GAA
ND5
R
R
6287–8003
6326–8057
1717
1732
0
0
ATT
ATT
T-tRNAPhe
T-tRNAPhe
tRNAHis
R
R
8019–8083
8058–8122
65
65
15
0
8051–8053 GTG
8090–8092 GTG
ND4
R
R
8084–9422
8123–9461
1339
1339
0
0
ATG
ATG
T-tRNAHis
T-tRNAHis
ND4L
R
R
9422–9709
9464–9754
288
291
−1
2
ATG
ATG
TAA
TAG
tRNAThr
F
F
9712–9775
9757–9821
64
65
2
2
9742–9744 TGT
9788–9790 TGT
tRNAPro
R
R
9776–9839
9822–9886
64
65
0
0
9808–9810 TGG
9854–9856 TGG
ND6
F
F
9842–10366
9889–10419
525
531
2
2
ATA
ATT
TAA
TAA
CytB
F
F
10371–11,519
10419–11,567
1149
1149
4
−1
ATG
ATG
TAA
TAA
tRNASer(UCN)
F
F
11,518–11,582
11,570–11,636
65
67
−2
2
11547–11549 TGA
11600–11602 TGA
ND1
R
R
11,603–12,544
11,654–12,592
942
939
20
17
ATG
ATG
TAA
TAA
tRNALeu(CUN)
R
R
12,546–12,612
12,594–12,660
67
67
1
1
12581–12583 TAG
12629–12631 TAG
lrRNA
R
R
12,613–13,926
12,661–13,995
1314
1335
0
0
tRNAVal
R
R
13,927–13,991
13,996–14,061
65
66
0
0
13961–13963 TAC
14029–14031 TAC
srRNA
R
R
13,992–14,767
14,062–14,836
776
775
0
0
D-loop
F
F
14,768–15,166
14,837–15,164
399
328
0
0
1* indicates the species ofEuploea mulciberand  2* indicates the species of Libythea celtis.Figure 1
Map of the mitogenomes ofEuploea mulciber and Libythea celtis. The abbreviations for the genes are as follows: ATP6, ATP8, ATP: synthase subunits 6 and 8 genes; CytB: cytochrome oxidase b gene; COI–COIII: cytochrome oxidase c subunit 1–3 genes; ND1–6, ND4L, NADH: dehydrogenase subunits 1–6 and 4L. Gene names are the standard abbreviations used in this paper; tRNA genes are denoted as one-letter symbol according to the IUPAC-IUB single letter amino acid codes. Gene names that are not underlined indicate the direction of transcription clockwise, while the underlining indicates counterclockwise.


## 3.2. Protein-Coding Genes

The total codon numbers of the 13 PCGs forEuploea 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, 36–38].Table 3
Nucleotide composition at each codon position of the concatenated 13 PCGs in the Papilionoidea mitogenome.
Species
1st  codon  position
2nd  codon  position
3rd  codon  position
Overall
A
T
C
G
A
T
C
G
A
T
C
G
A
T
C
G
Papilio xuthus
36.9
37.2
10.5
15.4
21.9
48.9
16.2
13.0
40.6
48.9
6.5
3.9
33.1
45.0
11.1
10.8
Agehana maraho
36.9
37.2
10.5
15.4
21.9
48.9
16.2
13.0
40.6
48.9
6.5
3.9
33.1
45.0
11.1
10.8
Troides aeacus
36.6
37.0
10.3
16.1
22.5
48.4
15.9
13.2
40.5
52.2
4.9
2.4
33.2
45.8
10.4
10.6
Sericinus montela
37.5
37.8
9.6
15.1
22.0
48.9
16.2
13.0
43.1
50.1
4.3
2.5
34.2
45.6
10.0
10.2
Teinopalpus aureus
37.0
37.0
10.4
15.6
21.9
48.2
16.6
13.3
40.6
50.2
5.5
3.7
33.1
45.1
10.8
10.9
Luehdorfia chinensis
36.6
37.4
10.1
15.9
22.2
48.5
16.2
13.1
42.7
51.3
3.7
2.3
33.8
45.7
10.0
10.4
Parnassius bremeri
37.0
37.9
9.4
15.7
22.2
48.4
16.4
13.0
44.1
51.0
3.3
1.7
34.4
45.8
9.7
10.2
Coreana raphaelis
38.3
38.1
8.8
14.9
22.0
49.0
15.8
13.2
45.8
51.2
2.0
1.0
35.4
46.1
8.8
9.7
Spindasis takanonis
38.6
38.2
9.2
14.1
22.5
49.4
15.2
12.9
43.6
51.0
2.8
2.7
34.9
46.2
9.1
9.9
Protantigius superans
37.7
37.7
9.2
15.3
22.0
48.8
15.9
13.2
44.0
50.6
3.2
2.2
34.6
45.7
9.4
10.2
Pieris rapae
36.0
37.6
9.9
16.5
22.1
48.0
16.6
13.4
42.7
48.2
5.3
3.8
33.6
44.6
10.6
11.2
Artogeia melete
36.7
36.8
10.3
16.2
21.9
47.9
16.8
13.4
42.3
49.8
4.6
3.4
33.6
44.8
10.6
11.0
Apatura ilia
37.2
37.6
9.6
15.6
22.0
48.8
15.9
13.2
41.7
49.7
5.3
3.3
33.6
45.4
10.3
10.7
Sasakia charonda
36.7
37.6
9.8
15.9
22.3
48.3
16.1
13.3
40.9
48.5
6.1
4.4
33.3
44.8
10.7
11.2
Sasakia
c
h
a
r
o
n
d
a
*
36.8
37.7
9.7
15.8
22.2
48.5
16.1
13.2
40.6
48.8
6.0
4.6
33.2
45.0
10.6
11.2
Kallima inachus
36.4
37.2
10.1
16.3
21.8
48.5
16.4
13.3
42.5
51.2
3.5
2.7
33.6
45.6
10.0
10.8
Argyreus hyperbius
37.4
37.1
9.9
15.6
22.6
48.2
6.1
13.1
41.5
51.5
4.6
2.4
33.8
45.6
10.2
10.4
Acraea issoria
36.6
36.7
10.7
15.9
23.0
47.8
16.2
13.1
39.7
50.1
6.4
3.7
33.1
44.9
11.1
10.9
Parathyma sulpitia
36.9
37.9
9.8
15.4
22.3
48.2
16.4
13.1
42.0
54.7
2.1
1.2
33.7
46.9
9.4
9.9
Calinaga davidis
36.3
37.7
10.4
15.7
22.2
48.4
16.4
13.1
40.7
51.5
5.1
2.7
33.0
45.8
10.6
10.5
Eumenis autonoe
35.9
36.3
11.3
16.4
21.5
48.4
16.4
13.7
39.7
48.8
7.1
4.4
32.3
44.5
11.6
11.5
Euploea mulciber
37.4
37.4
9.5
15.7
21.9
48.5
16.5
13.1
44.1
51.3
3.0
1.6
34.5
45.7
9.7
10.1
Libythea celtis
36.6
37.6
9.6
16.2
22.0
48.2
16.5
13.3
44.1
51.4
2.6
1.9
34.2
45.7
9.6
10.4
Ctenoptilum vasava
36.1
38.1
10.0
15.7
22.3
48.0
16.7
13.0
41.0
51.2
4.9
2.9
33.1
45.8
10.5
10.5
Start codons and stop codons were excluded in the count.*Sasakia charonda kuriyamaensis.Table 4
Codon usage of the protein-coding genes of theLibythea celtis and Euploea mulcibermitogenomes.
Codon
N (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)
N: frequency of codon used; RSCU: relative synonymous codon usage; *stop codon.
Start codons and stop codons were excluded in total codon counts.Table 5
Nucleotide composition and skewness in the rhopaloceran mitogenomes.
Major strand PCGs
Minor strand PCGs
Whole PCGs
Whole genome
Species
A + T
AT skew
GC skew
A + T
AT skew
GC skew
A + T
AT skew
GC skew
A + T
AT skew
GC skew
%
%
%
%
Papilio xuthus
77.1
-
0.114
-
0.206
79.8
-
0.210
0.334
78.1
-
0.152
-
0.014
80.0
-
0.011
-
0.216
Agehana maraho
77.1
-
0.114
-
0.206
79.8
-
0.210
0.334
78.1
-
0.152
-
0.014
80.6
0.006
-
0.262
Teinopalpus aureus
77.2
-
0.125
-
0.172
80.0
-
0.196
0.322
78.3
-
0.153
0.002
79.8
-
0.005
-
0.238
Troides aeacus
77.7
-
0.166
-
0.168
81.1
-
0.150
0.332
79.1
-
0.160
0.010
80.2
-
0.040
-
0.230
Parnassius bremeri
78.9
-
0.126
-
0.112
82.2
-
0.166
0.281
80.2
-
0.142
0.024
81.3
−0.011
−0.194
Sericinus montela
78.5
-
0.124
-
0.146
81.8
-
0.172
0.301
79.8
-
0.143
0.009
81.0
-
0.008
-
0.223
Luehdorfia chinensis
78.0
-
0.129
-
0.134
81.9
-
0.179
0.296
79.6
-
0.150
0.019
80.5
-
0.005
-
0.205
Ctenoptilum vasava
77.2
-
0.151
-
0.145
81.6
-
0.174
0.285
78.9
-
0.160
0.000
80.5
-
0.029
-
0.205
Pieris rapae
77.2
-
0.095
-
0.150
79.8
-
0.212
0.358
78.2
-
0.141
0.030
79.7
0.014
-
0.232
Artogeia melete
77.7
-
0.098
-
0.152
79.7
-
0.213
0.324
78.4
-
0.143
0.020
79.8
0.012
−0.222
Coreana raphaelis
80.3
-
0.143
-
0.074
83.4
-
0.115
0.270
81.5
-
0.132
0.044
82.7
-
0.047
-
0.158
Protantigius superans
79.3
-
0.137
-
0.087
81.9
-
0.140
0.276
80.3
-
0.138
0.041
81.7
-
0.036
-
0.174
Spindasis takanonis
80.1
-
0.104
-
0.117
82.7
-
0.193
0.338
81.1
-
0.139
0.044
82.4
0.004
-
0.214
Apatura ilia
77.9
-
0.124
-
0.140
80.7
-
0.187
0.314
79.0
-
0.149
0.021
80.4
-
0.011
-
0.207
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
c
h
a
r
o
n
d
a
*
77.2
-
0.121
-
0.142
79.7
-
0.196
0.324
78.2
-
0.151
0.028
79.9
-
0.007
-
0.218
Kallima inachus
78.1
-
0.136
-
0.125
81.1
-
0.177
0.336
79.2
-
0.152
0.036
80.3
-
0.013
-
0.212
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
-
0.124
-
0.094
81.8
-
0.174
0.297
79.9
-
0.144
0.040
81.2
-
0.017
-
0.181
Euploea mulciber
79.0
-
0.142
-
0.124
82.1
-
0.140
0.307
80.2
-
0.140
0.020
81.5
-
0.038
-
0.211
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 inEuploea 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 Figure2, 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 tRNATyr. 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 tRNATyr. 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].Figure 2
Alignment of the initiation context of the COI genes of 25 lepidopteran insect species, including that ofLibythea celtis and Euploea mulciber.The first several codons for COI and their amino acids are shown on the right-hand side of the figure. Wave-lined nucleotides indicate the adjacent partial sequence of tRNATyr. Arrows indicate the transcriptional direction. Boxed nucleotides indicate the currently proposed translation initiators for the COI gene of lepidopteran insects. The start codons for Libythea celtisand Euploea mulciberare designated as CGA. *Sasakia charonda kuriyamaensis.Nine of the PCGs inEuploea 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 theEuploea 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 tRNALeu (CUN) and tRNAVal genes [12, 13, 15, 16, 19, 37, 39–45]. 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 tRNAVal 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, bothEuploea 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 tRNASer (AGN), whose dihydrouridine (DHU) arm forms a simple loop (maps of secondary structure are not shown here). The case of tRNASer (AGN) with the simple loop in the DHU arm has also been detected in most of insect species including the lepidopterans determined till now [12–19, 36, 37, 39, 42, 45–48].


## 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. TheEuploea mulciber and Libythea celtis A + T-rich regions located between srRNA and tRNAMet were 399 and 328 bp in length, respectively (see Figure 1 and Table 2). Though the A + T-rich region ofLibythea 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 tRNAMet.Alignment of the initiation sites for the A + T-rich regions of 22 completely sequenced rhopaloceran mitogenomes is shown in Figure3, 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 inAgehana 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].Figure 3
Alignment of the initiation site for the A + T-rich regions of 23 completely sequenced lepidopteran mitogenomes. The shaded nucleotides indicate the poly-T stretch, and the boxed nucleotides indicate the conserved motif, ATAGA. *Sasakia charonda kuriyamaensis.A microsatellite-like elements (AT)3(TA)6 and (TA)12 preceded by the ATTTA motif are presented in the middle of theEuploea mulciber and Libythea celtis A + T-rich region, respectively. Similar cases are commonly found in all other sequenced lepidopteran species [12–15, 17, 19, 36–39, 42–44, 46–48]. Additionally, a 12 bp poly A interrupted by T and a 7 bp poly A interrupted by G presented immediately the upstream of the tRNAMet. 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 Figure4 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 totheCalinaga davidis(Calinaginae).NJ tree (a) and BI tree (b) of the main lineages of butterflies based on nucleotide sequences of the 13 protein-coding genes; numbers at the nodes: bootstrap values in (a) and posterior probabilities in (b).
(a)
(b)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, 55–57]. 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, 60–62]. 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.



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*Source: 491636-2013-02-21.xml*