Mitochondrial genomes of four satyrine butterflies and phylogenetic relationships of the family Nymphalidae (Lepidoptera: Papilionoidea)

Journal Pre-proof Mitochondrial genomes of four satyrine butterflies and phylogenetic relationships of the family Nymphalidae (Lepidoptera: Papilionoidea)

Mingsheng Yang, Lu Song, Lin Zhou, Yuxia Shi, Nan Song, Yalin Zhang PII:

S0141-8130(19)37136-3

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.008

Reference:

BIOMAC 14035

To appear in:

International Journal of Biological Macromolecules

Received date:

4 September 2019

Revised date:

1 December 2019

Accepted date:

2 December 2019

Please cite this article as: M. Yang, L. Song, L. Zhou, et al., Mitochondrial genomes of four satyrine butterflies and phylogenetic relationships of the family Nymphalidae (Lepidoptera: Papilionoidea), International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.12.008

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© 2019 Published by Elsevier.

Journal Pre-proof Mitochondrial genomes of four satyrine butterflies and phylogenetic relationships of the family Nymphalidae (Lepidoptera: Papilionoidea)

Mingsheng Yanga, b

a

Lu Songb

Lin Zhoua Yuxia Shib

Nan Songc

Yalin Zhanga

Key Laboratory of Plant Protection Resources and Pest Management of Ministry of

Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100,

College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, Henan,

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b

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China

466000, China;

College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450002,

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c

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*Correspondence:

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China.

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Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China. E-mail: [email protected]

Journal Pre-proof ABSTRACT The complete mitochondrial genomes (mitogenomes) of four Satyrini butterflies are newly determined and comparatively analyzed. These mitogenomes are all circular, double-stranded molecules, with the lengths of 15,194 bp (Minois dryas), 15,232 bp (Ypthima motschulskyi), 15,217 bp (Neope muirheadi) and 15,279 bp (Mycalesis francisca). Gene content and arrangement of newly sequenced mitogenomes are highly conserved and are typical of Lepidoptera. Interestingly, in M. francisca, a 48-bp insertion of macrosatellite (TA)24 is

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present at the trnE and trnF junction, which is rare in Lepidoptera. Among 13 protein-coding

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genes (PCGs) of reported Satyrinae mitogenomes, atp8 is a comparatively fast-evolving gene, and most PCGs of the four species sequenced show significant codon usage bias.

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Phylogenetic analyses based on the mitogenomes placed the four species sequenced in this

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study in Satyrini, confirming the result of morphological phylogeny. Moreover, phylogenetic

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analyses of the family Nymphalidae based on an expanded sampling and gene data from the GenBank and the present study show that several subtribe-level relationships in the speciose

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Satyrini are well supported as that previously defined by multiple-locus investigations.

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However, the subfamily-level relationships are not fully consistent across inference methods, and this needs further investigation based on mitogenome sequences of increased taxon sampling.

Keywords: Insect mitogenome; next-generation sequencing; Satyrinae

Journal Pre-proof 1. Introduction The Nymphalidae (brush-footed butterflies) is the most speciose family of butterflies with about 6,000 species distributed on all continents except Antarctica [1, 2]. Owing to species richness and ecological diversification, nymphalids have been intensely investigated as model taxa in ecological, conservation, evolutionary and developmental studies [3–5]. Regarding the classifications of Nymphalidae, twelve subfamilies have been defined, and their relationships have been inferred based on comprehensive data consisting of morphological characters and

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molecular sequences (e.g., Wahlberg et al. [6]) as well as phylogenomic dataset [7]. However,

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Nymphalidae phylogeny remains far from satisfactorily resolved mainly because of the existence of lowly supported or unstable nodes bearing some subfamilies. Satyrinae and

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Satyrini, including about 2,800 and 2,200 species, respectively, are the most diverse subfamily

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and tribe in Nymphalidae. From the Satyrinae and Satyrini, nine tribes (including Satyrini)

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and 13 subtribes were delimited respectively [2, 6, 8–10, 11]. However, phylogeny among them remains largely unresolved [2, 6, 8, 10, 11], and the key factor responsible for this is

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[10, 11].

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regarded as the long branch attraction with respect to rapid radiation evolution of this group

The mitochondrial genome (mitogenome) is a circular, double-stranded molecule that usually encodes 37 genes, including 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an A + T-rich region [12]. Due to cellular abundance, an absence of introns, rapid evolution, and a lack of extensive recombination, mitochondrial sequence can be easily amplified. Moreover, in recent years, the number of animal mitochondrial genome has been increasing following the development of sequencing technology, which in parallel provided effective data for studies on systematic, population genetics and evolutionary biology (reviewed by Cameron [13]; e.g., Timmermans et al. [14]; Song et al. [15]; Li et al. [16]; Nie et al. [17]; Song et al. [18]). In recent years, several mitogenome-based investigations focusing on Nymphalidae phylogeny were also performed. One landmark study was conducted by Wu et al. [19]. In this

Journal Pre-proof study, 30 nymphalid mitogenomes were sequenced and a phylogenetic analysis was performed based on these mitogenomes together with others on GenBank. The results on phylogenetic pattern of ten nymphalid subfamilies involved effectively conformed previous morphological, multiple-locus and phylogenomic studies [6, 7, 20]. A difference is that the basal group of Nymphalidae is Danainae in Wu et al. [19] rather than Libytheinae or Libytheinae plus Danainae recovered by the latter studies [6, 7, 20]. Recently, similar subfamily-level relationships (including the basal position of Danainae) with that of Wu et al.

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[19] were recovered by other two mitogenomic studies [21, 22]. However, the relationships

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among the Apaturinae, Biblidinae and Cyrestinae were inconsistent between the two studies, and the Biblidinae and Cyrestinae were not sampled in Wu et al. [19].

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In previous mitogenome-based investigations, only partial genome sequences or limited

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taxon sampling associated with Nymphalidae including the Satyrinae were conducted.

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Therefore, a comprehensive phylogenetic investigation on this family is needed based on denser taxon and mitochondrial sequence coverages. Satyrinae is the most diverse group of

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Nymphalidae but mitogenomes of only 18 species were sequenced (Table 1). In the present

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study, we newly determined and comparatively analyzed the complete mitogenomes of four Satyrini species: Minois dryas, Ypthima motschulskyi, Neope muirheadi and Mycalesis francisca. Besides, a comprehensive phylogenetic investigation on Nymphalidae was performed with expanded mitochondrial gene data and taxon sampling (70 species representing all 64 nymphalid genera with mitogenomes available). The aims of this study are to use all the available satyrine mitogenomes reported to improve the understanding of Satyrinae and Nymphalidae phylogeny.

2. Materials and methods 2.1. Sample collection, identification and DNA extraction Adult specimens were sampled at the Jigongshan mountain range, Henan Province, China, in June 2018. Specimens were identified according to morphological descriptions and

Journal Pre-proof illustrations (especially the genitalia) in Chou [31, 32]. Thorax muscle tissues were isolated to extract genomic DNA using the DNeasy tissue kit (Qiagen, Hilden, Germany). Voucher specimens are deposited in the Biology Laboratory of Zhoukou Normal University, China. 2.2. Mitogenome sequencing, assembly, annotation and sequence analysis Raw mitogenome sequences of four Satyrinae species were obtained by next-generation sequencing. After the exacted total DNA was quantified, Whole Genome Shotgun method was used to construct a library using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina,

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United States). Then, Illumina Miseq platform was employed for sequencing with a strategy

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of 250 paired-ends. FastQC was used for quality control. After processing with AdapterRemoval v. 2 [33] and SOAPec v. 2.01 [34], raw paired reads were filtered into

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high-quality reads. Then, A5-miseq v. 20150522 [35] and SPAdes v. 3.9.0 [36] were employed

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in de novo assembly, generating contig and scaffold sequences. Lastly, mitochondrial

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sequences were identified using BLASTn method, and MUMmer v. 3.1 [37] was used to establish position relationships among contig sequences and to fill in possible gaps.

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MITOS webserver was employed to annotate the complete mitogenome sequence with

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invertebrate genetic code [38]. Furthermore, tRNAScan-SE server v. 1.21 [39] was used to reidentify the 22 tRNAs, as well as to reconfirm their secondary structures. MEGA v. 6.06 [40] was used to reconfirm gene boundaries by aligning the newly sequenced mitogenome with previously reported satyrine mitogenomes. In the A + T-rich region, Tandem Repeats Finder program (http://tandem.bu.edu/trf/trf.html) [41] was used to detect possible tandem repeat elements. Strand asymmetry was calculated according to the following formulas: AT skew = [A – T]/[A + T] and GC skew = [G –C]/[G + C] [42]. Base composition and relative synonymous codon usage (RSCU) were calculated using MEGA v. 6.06 [40]. Nucleotide diversity and the ratio of nonsynonymous substitution (Ka) to synonymous substitution (Ks) for all PCGs were calculated using DNASP v. 5.0 [43]. In addition, effective codon usage statistics (Nc) was measured using CodonW 1.4.2 [44].

Journal Pre-proof 2.3. Phylogenetic analyses Phylogenetic analyses were performed based on extensive taxon sampling consisting of all 64 nymphalid genera and all 22 species of 16 Satyrinae genera with mitogenome sequenced. Accordingly, the ingroup included up to 70 nymphalids, and three Lycaenidae species were selected for outgroup taxa (Table S1) because the Lycaenidae was recovered as sister to the Nymphalidae by Wu et al. [19]. Among the mitochondrial genes, the 13 PCGs have been widely used in previous mitogenomic studies, and the rRNA genes might also be

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informative in resolving Satyrinae phylogeny demonstrated by our previous studies [45, 46].

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Therefore, two datasets were compiled in this study: one dataset consisting of all 37 mitochondrial genes (PCG-rRNA-tRNA) and the other consisting of the 13 PCGs and two

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rRNAs (PCG-rRNA). Sequence alignments were performed within the TranslatorX online

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platform [47] for 13 PCGs, and within the MAFFT online platform under the Q-INS-i

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algorithm [48] for two rRNAs and 22 tRNAs. Nucleotide sequence substitution model was selected using PartitionFinder v. 1.1.1 [49], with the Baysian Information Criterion (BIC)

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algorithm under a greedy search. The best partition scheme and corresponded nucleotide

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substitution models are shown in Table S2. Maximum likelihood (ML) analysis was performed using the IQ-TREE 1.6.7.1 [50] with the model determined by PartitionFinder. Bootstrap support (BS) was assessed using 1,000 ultrafast bootstrap replicates. Bayesian inference (BI) analysis was performed using MrBayes 3.1.2 [51] based on the same model as used in ML analysis. In this analysis, two independent Markov chain Monte Carlo (MCMC) runs were performed for 10,000,000 generations sampling per 100 generations. The convergence between the two runs was established by the Tracer version 1.6 (Effective sample sizes > 200) [52]. After the first 25% of the yielded trees were discarded as burn-in, a 50% majority-rule consensus tree with the posterior probability (PP) was generated from the remaining trees.

Journal Pre-proof 3. Results and discussion 3.1. General features of the newly sequenced mitogenomes The four newly sequenced mitogenomes are all circular, double-stranded molecules, and the lengths are 15,194 bp (M. dryas), 15,232 bp (Y. motschulskyi), 15,217 bp (N. muirheadi) and 15,279 bp (M. francisca) (Table 2–3). The lengths are comparable to other completely sequenced Satyrinae mitogenomes, which range from 15,122 bp in Melanitis leda to 15,721 bp in Stichophthalma louisa. Gene content and arrangement of the four Satyrinae

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mitogenomes are highly conserved and typical of Lepidoptera. The typical 37 mitochondrial

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genes (13 PCGs, 22 tRNAs, and two rRNAs) and an A + T-rich region are included, 23 of

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which (nine PCGs and 14 tRNAs) are encoded on the majority strand (J-strand), and the remaining genes are located on the minority strand (N-strand). Interestingly, in Lethe

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albolineata mitogenome [25], two tRNA-like pseudo-genes were recognized in rrnL gene and

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A + T-rich region, respectively. The existence of pseudo-gene in A + T-rich region was also reported in other butterflies such as Spindasis takanonis and Protantigius superans of

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Lycaenidae [53].

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Similar to other insect mitogenomes [12], A/T content in Satyrinae mitogenomes are highly biased, ranging from 79.9% in M. francisca to 81.8% in Y. motschulskyi (Table 3). AT skew and GC skew are routinely used to describe the base composition of mitogenomes [42, 54]. The negligible T skew and moderate C skew in newly sequenced mitogenomes are similar to other Lepidoptera and most insect species [55].

3.2. Protein-coding genes The total length of 13 PCGs of M. dryas, Y. motschulskyi, N. muirheadi and M. francisca are 11,225 bp, 11,215 bp, 11,230 bp and 11,228 bp, encoding 3,741, 3,738, 3,743 and 3,742 amino acids respectively (Table 3). In all sequenced mitogenomes, nine of the 13 PCGs are encoded on the J-strand, while the other four are located on the N-strand. The A + T content of

Journal Pre-proof the 13 PCGs varies from 78.1% in M. francisca to 80.5% in Y. motschulskyi. Regarding start and stop codons, most PCGs use the conventional ATN as the start codon (Table 2). However, in cox1, the unconventional CGA is consistently found. TAA is employed as stop codon in most PCGs, but the incomplete termination codon T is consistently used in cox2 of three species and nad4 of all four species. Incomplete termination codons are commonly recognized across arthropod mitogenomes, which may be related to post-transcriptional modification during the mRNA maturation process [56].

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To characterize evolutionary pattern of 13 PCGs, nucleotide diversity and the ratio of

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Ka/Ks across all Satyrinae mitogenomes were calculated for each PCG aligned. As shown in Fig. 1, nad6 and atp8 show the highest nucleotide diversity, in contrast to cox1 with the value

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of nucleotide diversity being the lowest. The Ka/Ks value for atp8 is the highest, followed by

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the nad4l, nad5, nad6, and the lowest value is for cox1 as that of nucleotide diversity. Notably,

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Ka/Ks values for all PCGs are lower than one, indicating that they are evolving under purifying selection. From these analyses, we can conclude that the atp8 is under the least

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Satyrinae.

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selection pressure and the most fast-evolving gene amongst the mitochondrial PCGs in

To understand genetic codon bias of the newly sequenced mitogenomes, the RSCU and Nc were measured. As shown in Fig. 2, for most amino acids, the usages of synonymous codons are biased. Moreover, the synonymous codon preferences are conserved among the four species, as may ascribe to their close relationships belonging to the same butterfly tribe, and these preferences were also recognized in some other lepidopterans [57, 58]. In detail, the three most used codons for the four species sequenced are consistently AUU, UUA and UUU. These codons encode Ile, Leu and Phe which are also the frequently used amino acids in these species. The codon usage bias was further evaluated by the Nc values. The Nc values are routinely regarded between 20–61, and are negatively correlated with codon usage bias. The Nc value = 20 indicates absolute bias toward a synonymous codon whereas Nc = 61 indicates the neutral codon usage [59]. Nc values for our analyses ranged from 30.1 to 33.78 indicating

Journal Pre-proof some trends of codon usage bias among the four species sequenced.

3.3. Transfer and ribosomal RNA genes For all newly sequenced Satyrinae mitogenomes, the typical 22 tRNAs are expectedly recognized (Table 2). Among them, eight tRNAs are encoded by the N-strand and the remaining 14 by the J-strand. The lengths are from 62 bp (trnR) to 71 bp (trnK) in M. dryas, Y. motschulskyi and M. francisca, from 60 bp (trnS1) to 71 bp (trnK) in N. muirheadi. As shown

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in Fig. 3, all tRNAs exhibit typical clover-leaf secondary structure, but trnS1 (AGN) lacks the

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DHU arm, a feature generally present in all Lepidoptera insects as well as in other metazoan mitogenomes [60, 61]. In four sequenced mitogenomes, 23, 18, 23 and 27 unmatched base

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pairs are present in M. dryas, Y. motschulskyi, N. muirheadi and M. francisca respectively,

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most of which in each species are the overrepresented noncanonical G-U pair. This

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overrepresentation is commonly present in tRNAs of insect mitogenomes [62–65]. Further, comparative tRNA analyses among four newly sequenced mitogenomes found that substantial

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nucleotide variation exists in several tRNAs, and most of these variations occurred in the

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DHU loops, TψC arms and TψC loops (Fig. 3). Two rRNA genes, rrnS and rrnL, were recognized in all newly sequenced mitogenomes, and they are located between trnV and the A + T-rich region and between trnV and trnL1 respectively (Table 2). The rrnS lengths range from 772 bp in N. muirheadi to 815 bp in Y. motschulskyi; and the rrnL lengths are from 1,333 bp in N. muirheadi to 1,341 bp in M. francisca.

3.4. Gene overlapping and intergenic regions Four gene-overlapping regions (with the number of nucleotide > 4) are conserved and consistently present in four newly sequenced mitogenomes (Table 2; Fig. 4). One is composed of the “AAGCCTTA” at the trnW and trnC junction (Fig. 4A); the second is a shorter sequence of “TCTAA” locating at the cox1 and trnL2 junction (Fig. 4C). The

Journal Pre-proof gene-overlapping region between atp8 and atp6, a 7-bp motif of “ATGATAA”, is regarded as a common feature in Lepidoptera even all insects (Fig. 4B). The fourth gene-overlapping regions at the trnF and nad5 junctions across four mitogenomes are not completely identical in nucleotide composition (Fig. 4D). The one in Y. motschulskyi is longer than that of other three species. In addition to the A + T-rich region, 84, 119, 76 and 124 intergenic nucleotides were recognized in M. dryas, Y. motschulskyi, N. muirheadi and M. francisca respectively. The long

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intergenic regions, which include 45–53 intergenic nucleotides, were expectedly detected and

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consistently present between trnQ and nad2 (Fig. 4E). This region, characterized by high A/T content, is widely present in Lepidoptera and even be regarded as a synapomorphy of

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lepidopteran species [66, 67]. The intergenic region at trnS2 and nad1 junction has been

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widely reported in insect mitogenomes as characterized by the existence of the motif

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“ATACTAA” responsible for mitochondrion transcription [55, 67, 68]. However, our study reveals that the motif “ATACTAA” is putatively located in intergenic region of only Y.

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motschulskyi, contrasting to at 3′ end of the nad1 for other three species (Fig. 4F).

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Accordingly, the predicted nad1 length of the Y. motschulskyi is shorter than that of other three species. However, this result needs to be further confirmed based on the nad1 mRNA expression data [65, 69], mainly because that the 3’ end of nad1 was predicted only by the position of routinely used stop codon “TAA”. In addition, an interesting result regarding on the intergenic region is that, at the trnE and trnF junction, a 48-bp insertion of macrosatellite (TA)24 was detected only in M. francisca, which is rare in reported butterfly mitogenomes, to our knowledge. The A+T-rich regions of all four sequenced mitogenomes are located between rrnS and trnM (Table 4; Fig. 4G), and the A + T content of this region ranges from 92.7% in M. francisca to 94.6% in M. dryas. Insect mitochondrial A + T-rich region is usually structured by several conserved sequence blocks responsible for mitogenome replication and transcription [70]. These blocks include (from 5’ to 3’ end) the motif “ATAGA” and

Journal Pre-proof subsequent poly-T structure, the motif “ATTTA”, macrosatellite (AT)n or (TA)n element, and an “A”-rich 3’ end upstream of the trnM gene. Insect A + T-rich region is generally characterized by the presence of multiple tandem repeat elements [71]. However, in all four sequenced mitogenomes, no tandem repeat element was detected.

3.5. Phylogenetic analyses Two datasets based on the same inference method yielded identical topologies in terms

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of subfamily-level relationships. The relationships of five previously defined Nymphalidae

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clades obtained from the ML and BI analyses (Figs. 5–6) are mostly identical, but differences exist. In ML analyses, the danaine + (satyrine + (libytheine + (nymphaline + heliconline)))

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was recovered, greatly reinforcing that of previous mitogenomic studies focusing on the

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Nymphalidae [19, 21, 22]. In BI analyses, their relationships are libytheine + ((danaine +

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satyrine) + (nymphaline + heliconline)). In detail, the Libytheinae is basal to the rest Nymphalidae, which provides supports for several morphological, multiple-locus and

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phylogenomic studies [6, 7, 20] rather than mitogenomic studies [19, 21, 22]. Moreover, the

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sister danaine and satyrine was unexpectedly detected, but this relationship may be unstable because of the low posterior probabilities (< 0.95). In the nymphaline clade, the Nymphalinae + Heliconlinae is defined with strong supports by both ML and BI analyse, a result consistently recovered by other studies based on various data as well (e.g. Freitas and Brown [20]; Peña and Wahlberg [2]; Wahlberget et al. [6]; Wu et al. [19]; Shi et al. [21]; Liu et al. [22]; Espeland et al. [7]). The heliconline clade contains five subfamilies, and their relationships recovered herein are Pseudergolinae + (Nymphalinae + (Cyrestinae + (Apaturinae + Biblidinae))), which is completely identical to that of Liu et al. [22] based on 13 mitochondrial PCGs but showing a little difference with that of Shi et al. [21] employing 13 PCGs plus two rRNAs. In the latter study, the Cyrestinae, instead of Biblidinae, forms a sister group with Apaturinae. In addition, another proposal regarding the phylogeny of heliconline clade is present, that is, Cyrestinae first clusters with Nymphalinae, and then

Journal Pre-proof being sister to the Apaturinae + Biblidinae [6, 7]. The satyrine clade, including Calinaginae, Charaxinae and Satyrinae, is a well-defined group recovered by various studies [2, 6, 7, 19], and the Calinaginae is always recovered as sister to Charaxinae + Satyrinae excepting our ML analysis based on the dataset including 13 PCGs and two rRNAs. Collectively, our BI analyses firstly provide mitogenome evidence for morphological, multiple-locus and phylogenomic studies [6, 7, 20] in the phylogenetic position of Libytheinae in Nymphalidae. However, the results of our ML analyses are consistent with that of previous mitogenomic

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studies. This discrepancy may be resolved by more comprehensive mitogenome sampling in

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future study.

In Nymphalidae, Satyrinae is the most speciose subfamily. However, its phylogenetic

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relationships at both tribe and subtribe levels remain largely unresolved [2, 6, 8, 45, 46]. With

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an attempt to better understand their evolutionary relationships, a preliminary investigation

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was performed firstly based on mitogenomic data herein. At the tribe level, our ML and BI analyses recover the relationships among the four satyrine tribes analyzed as (Amathusiini +

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Elymniini) + (Melanitini + Satyrini). The close Amathusiini and Elymniini is in accordance

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with the BI analysis of Peña and Wahlberg [2], but some studies including our previous investigation [6, 45] regarded the Elymniini more close to the Melanitini. Thus, the tribe level relationships obtained herein need further verification based on more sampling taxa. Satyrini represents the most diverse tribe (about 2,200 speices) in Satyrinae, and 13 subtribes are defined [11]. In the present study, mitogenomes of four Satyrini species are newly sequenced, which increases the number of reported Satyrini mitogenomes to 17. Although the taxon sampling is still limited for the large Satyrini in species composition, the close relationships among some subtribes such as the grouping of Parargina, Lethina and Mycalesina, and the grouping of Melanargiina, Satyrina and Ypthimina, as recovered in our previous study [46] and other multiple-locus investigations [2, 6, 10], are well supported especially in our BI analysis. However, it should be noted that only seven of the 13 subtribes are analyzed in the present study, and further effort is needed to improve the understanding of

Journal Pre-proof the whole Satyrini phylogeny based on mitogenome sequences of increased sampling. Selecting suitable genetic markers is of great importance in studies of molecular systematics. Among mitochondrial genes, the 13 PCGs, possibly being easily alignable and without secondary structure, have been widely used and proven informative in insect systematics. Furthermore, our previous studies [45, 46] have verified that the rRNA genes were also informative for phylogenetic analysis of the Satyrinae. In the present study, to test the effectiveness of tRNAs in reconstructing Nymphalidae phylogeny, two datasets were

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compiled: all 37 mitochondrial genes, and the 13 PCGs plus two rRNAs. Our results show

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that the inclusion of tRNAs, based the same inference method, generally not affect the topology. However, the supports of most nodes especially in ML analysis are increased,

Conflicts of Interest

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indicating that tRNAs contribute positively to the nodes related in the present analyses.

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All authors have read and approved the final manuscript. The authors declare no conflict

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of interest.

Acknowledgements

This work was funded by the Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education of China (A115020002), the National Natural Science Foundation of China (31702046), and the Project of Scientific Research Innovation Fund for College Student (ZKNUD2019019 and ZKNUD2019076).

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Journal Pre-proof Table 1 The Satyrinae species with available mitogenome on GenBank. Taxa

Mitogenome size (bp)

GenBank accession no.

Reference

15,721

KP247523

Unpublished

14,020

KF990129

Shi et al. [21]

15,167

KF906484

Shi et al. [21]

Melanitis phedima

15,142

KF590538

Wu et al. [19]

Melanitis leda

15,122

JF905446

Shi et al., [23]

Satyrinae Amathusiini Stichophthalma louisa Stichophthalma howqua



Elymniini Elymnias hypermnestra

of

Melanitini

Satyrini 15,240

Lasiommata deidamia

15,244

Lethina Lethe dura

15,259

Neope pulaha

15,209

Neope muirheadi Ninguta schrenckii

da Costa [24]

MG880214

Unpublished

KF881051

Li et al. [25]

KF906485

Shi et al. [21]

KF590543

Wu et al. [19]

15,217

MN242789

This study

15,261

KF881052

Fan et al. [26]

15,267

KM244676

Tang et al. [27]

15,279

MN242790

This study

15,143

KF906487

Zhang et al. [28]

15,142

KF906486

Huang et al. [29]

Davidina armandi

15,214

KF881046

Unpublished

Hipparchia autonoe

15,489

GQ868707

Kim et al. [30]

Minois dryas

15,194

MN242787

This study

Ypthima akragas

15,227

KF590553

Wu et al. [19]

Ypthima motschulskyi

15,232

MN242788

This study

Callerebia suroia

15,208

KF906483

Unpublished

Mycalesis mineus Mycalesis francisca

Jo ur

Coenonymphina

Triphysa phryne Melanargiina

Melanargia asiatica Satyrina

na

Mycalesina

re

15,248

lP

Lethe albolineata

KJ547676

-p

Pararge aegeria

ro

Parargina

Ypthimina

Note: * the mitochondrial genome of the indicated species is incomplete

Journal Pre-proof Table 2 The four newly determined Satyrinae mitogenomes. Position

Anticodon

Intergenic nucleotides

67/67/68/66

CAT

0/-1/0/1

68/67/69/68

132/132/136/134

GAT

-3/-3/-3/-3

N

130/130/134/132

198/198/202/200

TTG

53/51/45/51

nad2

J

252/250/248/252

1,265/1,263/1,261/1,265

trnW

J

1,264/1,262/1,260/1,264

1,330/1,330/1,326/1,330

trnC

N

1,323/1,323/1,319/1,323

1,386/1,387/1,381/1,386

trnY

N

1,387/1,387/1,382/1,386

1,451/1,450/1,444/1,452

cox1

J

1,457/1,454/1,447/1,458

2,992/2,989/2,982/2,993

trnL2

J

2,988/2,985/2,978/2,989

3,054/3,051/3,044/3,055

cox2

J

3,055/3,052/3,046/3,056

3,733/3,726/3,724/3,734

trnK

J

3,731/3,728/3,722/3,732

3,801/3,798/3,792/3,802

trnD

J

3,803/3,805/3,794/3,803

3,868/3,873/3,859/3,869

atp8

J

3,869/3,874/3,860/3,870

4,030/4,038/4,018/4,031

ATT/ATT/ATC/ATC

TAA/TAA/TAA/TAA

-7/-7/-7/-7

atp6

J

4,024/4,032/4,012/4,025

4,701/4,709/4,689/4,702

ATG/ATG/ATG/ATG

TAA/TAA/TAA/TAA

0/-1/3/-1

cox3

J

4,702/4,709/4,693/4,702

5,490/5,497/5,481/5,490

ATG/ATG/ATG/ATG

TAA/TAA/TAA/TAA

2/9/2/2

trnG

J

5,493/5,507/5,484/5,493

5,559/5,572/5,549/5,558

nad3

J

5,560/5,573/5,550/5,559

5,913/5,926/5,903/5,912

trnA

J

5,915/5,929/5,905/5,912

5,979/5,993/5,970/5,979

TGC

1/-1/1/4

trnR

J

5,981/5,993/5,972/5,984

6,042/6,054/6,034/6,045

TCG

4/1/2/6

trnN

J

6,047/6,056/6,037/6,052

6,113/6,121/6,103/6,117

GTT

-3/-3/-3/-3

trnS1

J

6,111/6,119/6,101/6,115

6,170/6,178/6,160/6,174

GCT

1/1/8/1

trnE

J

6,172/6,180/6,169/6,176

6,236/6,246/6,233/6,247

TTC

-2/3/-2/48

trnF

N

6,235/6,250/6,232/6,296

6,300/6,315/6,299/6,361

GAA

-17/-29/-17/-17

Feature

Strand

trnM

From

To

J

1/1/1/1

trnI

J

trnQ

Stop codon

f o

ATT/ATT/ATT/ATT

o r p

e

r P

rn

u o

J

l a

Initiation codon

CGA/CGA/CGA/CGA

ATG/ATG/ATG/ATG

TAA/TAA/TAA/TAA

-2/-2/-2/-2 TCA

-8/-8/-8/-8

GCA

0/-1/0/-1

GTA

5/3/2/5

TAA/TAA/TAA/TAA

-5/-5/-5/-5 TAA

T/TAA/T/T

-3/1/-3/-3 CTT

1/6/1/0

GTC

0/0/0/0

TCC ATT/ATT/ATT/ATT

0/0/1/0

TAA/TAA/TAA/TAA

0/0/0/0 1/2/1/-1

Journal Pre-proof nad5

N

6,284/6,287/6,283/6,345

8,035/8,053/8,037/8,099

trnH

N

8,036/8,051/8,035/8,097

8,101/8,117/8,098/8,163

nad4

N

8,105/8,121/8,102/8,167

9,440/9,456/9,437/9,502

ATG/ATG/ATG/ATG

T/T/T/T

-1/1/-1/-1

nad4l

N

9,440/9,458/9,437/9,502

9,727/9,745/9,727/9,789

ATG/ATG/ATG/ATG

TAA/TAA/TAA/TAA

2/2/2/3

trnT

J

9,730/9,748/9,730/9,793

9,793/9,811/9,794/9,856

TGT

0/0/0/0

trnP

N

9,794/9,812/9,795/9,857

9,858/9,876/9,859/9,922

TGG

2/2/2/-2

nad6

J

9,861/9,879/9,862/9,925

10,388/10,403/10,395/10,449

ATC/ATT/ATC/ATA

cob

J

10,388/10,403/10,395/10,449

11,539/11,554/11,546/11,603

ATG/ATG/ATG/ATG

trnS2

J

11,548/11,553/11,545/11,602

11,612/11,618/11,612/11,667

nad1

N

11,614/11,653/11,612/11,666

12,570/12,588/12,565/12,622

trnL1

N

12,572/12,590/12,569/12,623

12,638/12,659/12,636/12,689

rrnL

N

12,639/12,635/12,637/12,690

13,976/13,995/13,969/14,030

trnV

N

13,977/13,994/13,970/14,031

14,040/14,058/14,032/14,094

rrnS

N

14,041/14,058/14,033/14,095

14,819/14,871/14,804/14,869

14,820/14,872/14,805/14,870

15,194/15,232/15,217/15,279

A+T-rich region

n r u

l a

ATT/ATG/ATT/ATT

TAG/TAA/TAA/TAA

0/-3/-3/-3 GTG

o r p

ATA/ATG/ATA/ATG

e

r P

f o

TAA/TAA/TAA/TAA

-1/-1/0/-1

TAA/TAA/TAA/TAA

8/-2/-2/-2 TGA

TAA/TAA/TAA/TAA

o J

1/34/-1/-2 1/1/3/0

TAG

0/0/0/0 0/0/0/0

TAC

Note: “J” indicates the majority strand and “N” indicates the minority strand; The characters divided by the “/” correspond to the M. dryas/Y. motschulskyi/N. muirheadi/M. francisca.

3/3/3/3

0/0/0/0 0/0/0/0

Journal Pre-proof Table 3 Nucleotide composition of the four newly determined Satyrinae mitogenomes. Size (bp)

AT content (%)

AT-skew

GC-skew

Genome

15,194/15,232/15,217/15,279

80.2/81.8/80/79.9

-0.026/-0.051/-0.055/-0.036

-0.222/-0.209/-0.236/-0.214

PCGs

11,225/11,215/11,230/11,228

78.6/80.5/78.5/78.1

-0.16/-0.15/-0.157/-0.16

-0.009/0.026/-0.009/0.014

tRNAs

1,444/1,454/1,450/1,459

80.9/81.8/80.7/81.3

0.028/0.017/0.016/0.031

0.173/0.127/0.161/0.176

rRNAs

2,117/2,175/2,105/2,116

85.2/85.9/84.7/85.3

0.086/0.099/0.096/0.086

0.347/0.348/0.359/0.329

A+T-rich region

315/361/413/410

94.6/93.9/93/92.7

0.021/-0.086/-0.088/-0.115

-0.296/-0.377/-0.029/0

f o

o r p

Note: The characters divided by the “/” correspond to the M. dryas/Y. motschulskyi/N. muirheadi/M. francisca.

l a

o J

n r u

r P

e

Journal Pre-proof Figure legends:

Fig. 1. Nucleotide diversity (A) and the ratio of Ka/Ks (B) of PCGs from 22 reported Satyrinae mitogenomes. Fig. 2. Relative synonymous codon usages (RSCU) in PCGs of four newly determined mitogenomes. Codon families are indicated below the X-axis. Fig. 3. Putative secondary structures of tRNAs from Y. motschulskyi mitogenome. The tRNAs are labeled with the abbreviations of their corresponding amino acids. Dashes indicate the

of

Watson-Crick base pairs; dots indicate the wobble GU pairs; and the other non-canonical pairs

ro

are not marked. The nucleotides variable among four newly sequenced mitogenomes is

-p

marked.

re

Fig. 4. Gene overlapping and intergenic regions among four sequenced Satyrinae mitogenomes. Nucleotides colored red indicate the sequence of overlapping or intergenic

lP

regions unless where is further explanatory. A. The overlapping region between the trnW and

na

trnC. B. The overlapping region between the atp8 and atp6. C. The overlapping region between the cox1 and trnL2. D. The overlapping region between the trnF and nad5. E. The

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intergenic region between trnQ and nad2. F. The intergenic sequence between trnS2 and nad1, and the motif “ATACTAA” routinely found in insect mitogenomes is colored red. G. Schematic illustration of the A + T-rich region from four newly sequenced mitogenomes. The conserved motifs ATAGA and ATTTA were colored green. Dots indicate omitted sequences, and the number of dot is not proportional to nucleotide number of corresponding part. Fig. 5. ML trees inferred from IQ-TREE analyses. The species with newly sequenced mitogenome are emphasized in bold. Numbers separated by slash (/) on node represent the bootstrap supports based on the PCG-rRNA-tRNA and PCG-rRNA datasets respectively. The “-” represents unrecovered node based on PCG-rRNA dataset. Fig. 6. BI trees inferred from MrBayes analyses. The species with newly sequenced mitogenome are emphasized in bold. Numbers separated by slash (/) on node represent the

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