The first mitochondrial genome for the butterfly family Riodinidae(Abisara fylloides) and its systematic implications

Fang ZHAO, Dun-Yuan HUANG,2, Xiao-Yan SUN, Qing-Hui SHI, Jia-Sheng HAO,*, Lan-Lan ZHANG, Qun YANG,*

1. College of Life Sciences, Anhui Normal University, Wuhu 24100, China

2. College of Forestry, Jiangxi Environmental Engineering Vocational College, Ganzhou 34100, China

3. Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing 21008, China

The typical metazoan mitochondrial genome(mitogenome) contains 37 genes, including 13 proteincoding genes (PCGs), 2 rRNA genes and 22 tRNA genes,and a non-coding area (i.e., the control region or the A+T-rich region) (Wolstenholme, 1992; Boore, 1999).Maternal inheritance, lack of recombination and an accelerated evolutionary rate compared with the nuclear genome have all contributed to the increased use of mitogenomes, which is one of the key methods in fields such as phylogenetics, comparative and evolutionary genomics, molecular evolution and population genetics(Ballard & Whitlock, 2004; Simonsen et al, 2006). At present, mitochondrial genomes have already been determined in a variety of insect groups covering nearly 200 species. However, reported complete mitogenomes are relatively scarce for lepidopterans and especially for butterflies. To our knowledge, as of October 2012 only about 20 butterfly species covering five butterfly families(Table 2) have been reported or deposited into the GenBank, but only one butterfly family, the Riodinidae,still lack corresponding data.

The phylogenetic position and taxonomic ranking of the butterfly family Riodinidae among butterfly lineages are still controversial issues among entomologists. Some scholars suggest that the riodinids are closely related to the lycaenids,1considering the similarities in morpholo gical character, behavior, and host plants between the two (sluglike larvae, pupa contigua, ants associated) (Ackery, 1984;Chou, 1998; de Jong et al, 1996; Ehrlich, 1958; Scott,1985). Moreover, they are usually classified into the Lycaenidae family as a subfamilial taxon. Some consider the riodinids a unique family parallel to the Lycaenidae family (Campbell et al, 2000; de Jong et al, 1996;Kristensen, 1976; Shou et al, 2006), and others propose that the riodinids are more closely related to the nymphalids than to the other butterfly groups, such as the lycaenids (Martin & Pashley, 1992; Robbins, 1987, 1988).

This study sequenced the first complete mitochondrial genome of the Abisara fylloides, a representative species of the family Riodinidae, by long PCR and primer walking techniques. Its genetic structure was preliminarily compared with those of other available butterfly species. The maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees of the A.fylloides and other available butterfly representative species were reconstructed based on concatenated DNA sequences of the 13 protein-coding genes (PCGs), and aim to clarify their phylogenetic relationships and further provide new information about the structure, organization and molecular evolution of the lepidopteran mitogenomes.

MATERIALS AND METHODS

Sample collection and DNA extraction

Adult individuals of A. fylloides were collected in Jinghong, Yunnan Province, China in August 2006. After collection, sample specimens were preserved in 100%ethanol immediately and stored at -20 °C before DNA extraction (specimen No. ZHF07). Total Genomic DNA was isolated using the proteinase K-SiO2method as described by Hao et al (2005).

PCR amplification and sequence determining

The multiple sequence alignments were conducted using the software Clustal X 1.8 based on the mitogenome sequences of Coreana raphaelis, Artogeia melete, Troides aeacus available from GenBank and those of Argyreus hyperbius, Acraea issoria, Calinaga davidis, Pieris rapae determined in our laboratory (Thompson et al, 1997). The long PCR primers, which may cover the whole mitogenome, were designed according to the conserved regions by the software Primer premier 5.0 (Singh et al,1998) (Table 1). Seven short fragment sequences (500-700 bp) of cox1, cox2, cox3, cytb, nad1, rrnL and rrnS were amplified using insect universal primers (Caterino &Sperling, 1999; Simmons & Weller, 2001; Simon et al,1994). All the primers were synthesized by the Shanghai Sheng gong Biotechnology Co. Ltd.

Table 1 List of PCR primers used in this study

Seven partial gene sequences were initially sequenced under the following conditions: an initial denaturation at 94 °C for 5 minutes, then denaturation at 94 °C for 1 minute for a total of 35 cycles; annealing at 45-55 °C for 1 minute and extension at 72 °C for 2 minutes plus 30 seconds; final extension at 72 °C for 10 minutes. Long PCRs were performed using TaKaRa LA Taq polymerase with the following cycling parameters:an initial denaturation for 5 minutes at 95 °C; followed by 30 cycles at 95 °C for 55 seconds, 45-55 °C for 2 minutes, 68 °C for 2 min and 30 seconds; and a subsequent final extension step of 68 °C for 10 minutes.

The PCR products were separated by electrophoresis in a 1.2% agarose gel and purified using the DNA gel extraction kit (TaKaRa). All PCR fragments were sequenced directly after purification with the QIA quick PCR Purification Kit reagents (QIAGEN). Internal primers were applied to complete sequences by primer walking(detailed primer information will be provided upon request).All fragments were sequenced for both strands.

Data analysis

We used DNASIS MAX (Hitachi) for sequence assembly and annotation. Protein-coding genes and rRNA genes were identified by sequence comparison with other available insect mitochondrial sequences. The tRNAs were identified by tRNAscan-SE v.1.21 (Lowe &Eddy, 1997). The putative tRNAs, which were not found by tRNAscan-SE, were identified by a sequence comparison of A. fylloides with the other lepidopteran tRNAs. PCGs were aligned with the other available lepidopteran mitogenomes using DAMBE software (Xia& Xie, 2001). The tandem repeats in the A+T-rich region were predicted using the Tandem Repeats Finder online(http://tandem.bu.edu/trf/trf.html) (Benson, 1999).Nucleotide composition was calculated using PAUP 4.0b10 (Swofford, 2002). The mitogenome sequence data have been deposited in GenBank under the accession number HQ259069.

Phylogenetic analysis

Phylogenetic analyses were performed on 19 representative species including A. fylloides, covering all the six families of butterflies. The multiple aligning of the concatenated nucleotide sequences of the 13 mitochondrial PCGs of the 19 species (Table 2) was conducted using ClustalX 1.8. The phylogenetic trees were reconstructed with the maximum likelihood (ML)and Bayesian inference (BI) methods, using the moth species Adoxophyes honmai (GenBank accession number of mitogenome: NC014295) as the outgroup. In both phylogenetic analyses, the third codon position of all the sequences was excluded. The ML analyses were conducted in PAUP 4.0b10 by using TBR branch swapping (10 random addition sequences) as a search method. The model GTR+I+Γ was selected as the best fit model using Modeltest 3.06 (Posada & Crandall, 1998)under the AIC scores, and the bootstrap values of the ML tree were evaluated via the bootstrap test with 1 000 iterations. The Bayesian analysis was performed using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001) with the partitioned strategy (13 partitions: cox1, cox2, cox3, atp8,atp6, nad1, nad2, nad4, nad4L, nad5, nad6 and cytb), and the best substitution model for each partition was selected as in the ML analysis. The MCMC analyses(with random starting trees) were run with one cold and three heated chains simultaneously for 1,000,000 generations sampled every 100 generations with a burnin of 25% until the average standard deviation of split frequencies to be less than 0.01, which means that convergence was reached.

RESULTS AND DISCUSSION

Genome structure and organization

The complete mitogenome is 15 301 bp in length,encodes 37 genes in all. It contains 13 protein, 22 tRNA,2 rRNA genes and a non-coding high A+T content region(Figure 1, Table 3). Its structure and organization are identical to those of the majority of other lepidopterans(Bae et al, 2004; Cha et al, 2007; Cameron & Whiting,2008; Hao et al, 2012; Hong et al, 2008, 2009; Hu et al,2010; Kim et al, 2009b; Ji et al, 2012; Junqueira et al,2004; Wang et al, 2011), though a few lepidopterans,such as three Thitarodes species, were reported to possess the ancestral gene order trnI-trnQ-trnM instead of the trnM-trnI-trnQ (Cao et al, 2012) .

Eight overlapping sequences totaling 61 bp are located throughout the A. fylloides mitogenome, with size ranging from 2 to 35 bp, of which the longest (35 bp)is located between the cox2 and the tRNALysgenes. In addition, 17 intergenic spacers ranging from 1 to 45 bp in length are found in the mitogenome. Among these spacers, the longest is located between the tRNAGlnand nad2 genes, the other 16 spacers are scattered throughout the whole genome (Figure 1, Table 3). Most of these spacer regions are arranged relatively compactly compared with other insect mitogenomes (Cameron &Whiting, 2008; Hao et al, 2012; Hong et al, 2009; Hu et al, 2010; Kim et al, 2009b; Ji et al, 2012; Junqueira et al,2004; Wang et al, 2011).

Figure 1 Circular map of the mitochondrial genome of Abisara fylloides

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Table 3 Organization of the Abisara fylloides mitochondrial genome

The A. fylloides A+T-rich region is flanked on one side by the rrnS and on the other side by the tRNAMetgenes. This region exhibits the highest A+T content(91.0%) (Table 4), and spans 423 bp (Table 3, Table 4). A sequence analysis of the A+T-rich region revealed that it contained some structures typical of other lepidopteran mitogenomes: (1) at 20 bp downstream of the small subunit rRNA gene, there is a structure including a motif ‘ATAGA’ which is very well conserved in all sequenced lepidopteran insects, and a 18-bp polyT stretch, both of which have been suggested as the origin of minority or light strand replication (ON) and to play a regulatory role (Kim et al, 2009a; Lutz-Bonengel et al,2004; Saitou et al, 2005; Yukuhiro et al, 2002). (2)Between the sites 15 151 and 15 168 there is a microsatellite-like (TA)9element, which is also found in the majority of other lepidopterans; (3) There is another motif “ATTTA” of unknown function located from 15 139 to 15 143 upstream of the (TA)9, which is also typical of the other lepidopterans. In addition, to our great surprise, an unexpected short microsatellitelike repeating region (TA)11downstream of the (TA)9was detected in the A+T-rich region, and this has not been reported in any other lepidopterans.

Table 4 Nucleotide composition and skewness in different regions of the Abisara fylloides mitogenome

Base composition bias

The base composition of the A. fylloides mitogenome shows an A and T bias (Table 4). The whole A+T content of the mitogenome is up to 81.2%,ranging from that of Argyreus hyperbius (80.81%) to that of Coreana raphaelis (82.66%). Like most other metazoan mitogenomes, the A+T content of the A+T-rich region, which is located between the rrnS and tRNAMetgenes, is the highest (91.0%) in all known butterfly species except for P. rapae (Pieridae) to date.The base contents of A, T, C, G are 39.5%, 41.7%,11.3%, 7.5%, respectively, indicating a relatively higher A+T content (81.2%). These phenomena commonly exist in the protein-coding genes, which have a relatively lower A+T content (79.8%), and the tRNA and rRNA genes in insects.

Protein-coding genes

The sequences of the 13 Abisara fylloides PCGs are 11 224 bp in length, including 3 730 codons(excluding termination codons). Twelve of the 13 PCGs use standard ATN as their start codon except for the cox1 gene, and eight of these 12 PCGs begin with ATA or ATG (Methionine) and the other four begin with ATT or ATC (Isoleucine) (Table 3). The start codons for cox1 gene of lepidopteran insects have usually been a controversial issue. In general, there is no typical start codon, especially for the cox1 gene,which usually uses CAG (R) as start codon in insects,including the lepidopterans. However, some scholars reported unusual start codons, such as the trinucleotide TTG (Bae et al, 2004; Hong et al, 2008),ACG (Lutz-Bonengel et al, 2004), GCG (Nardi et al,2003), the tetranucleotide ATAA, ATCA and ATTA(Clary & Wolstenholme, 1983; de Bruijn,1983; Kim et al, 2006), and the hexanucleotides TATTAG (Flook et al, 1995), TTTTAG (Yukuhiro et al, 2002), TATCTA(Coates et al, 2005), ATTTAA (Beard et al, 1993;Mitchell et al, 1993) for cox1 in some other insect species. However, the CGA is present as a conserved region for all lepidopteran insects reported, and thus we tend to consider that CGA is the cox1 start codon for A. fylloides as Kim et al (2009b) suggested. As for stop codons, 9 of the 13 PCGs use standard TAA except for the cox1, cox2, nad4 and nad5 genes, all of which terminate at a single thymine (Table 3), and this case was also found in all other lepidopterans reported to date. For more details on this phenomenon, please refer to the discussions of Kim et al. (2010).

The PCG amino acid sequence variation analysis showed that there were 3 755 homologous sites, of which 1 964 are conserved, 1 791 are variable, and 1 128 are parsimony informative. Among the twenty amino acids in the 13 PCGs, six (Leu, Met, Ile, Phe,Asn, and Tyr) were used more frequently than the others, and their usage frequencies were higher than the average, whereas the other 14 amino acids less used (Table 5).

rRNA and tRNA genes

The large subunit rRNA and small subunit rRNA genes of the A. fylloides are 1 334 and 771 bp in length,respectively. As in other lepidopterans, these two genes are located between tRNALeu(UUR)and tRNAVal, and between tRNAValand A+T-rich region respectively(Cameron & Whiting, 2008; Hao et al, 2012; Hu et al,2010; Kim et al, 2009a; Salvato et al, 2008; Wang et al,2011; Yang et al, 2009).

The A. fylloides mitogenome harbors 22 tRNA genes, which are scattered throughout the whole genome and ranged in length from 61 to 71 bp. Except for the tRNASer(AGN), which lacks the DHU loop, all tRNAs are shown to be folded into the cloverleaf secondary structures, within which all amino acid acceptor stems have 7 base pairs, and all anticodon stems have 5 base pairs. This was also found in all the other lepidopterans determined to date (Cameron & Whiting, 2008; Hao et al,2012; Hu et al, 2010; Kim et al, 2009a).

A total of 27 pairs of base mismatches were detected in all the predicted tRNA secondary structures,among which 17 are GU, 6 are UU, 2 are AA, and 2 are AC. The AA mismatches occur at the anticodon stem of tRNALysand the TψC loop of tRNATrp; the AC mismatches occur at the amino acid acceptor stem of tRNATyrand the anti codon stem of tRNALeu(UUR). Among the 6 UU mismatches, two occur at the amino acid acceptor stems of tRNALeu(UUR)and tRNAAla, two at the anti codon stems of tRNALeu(UUR)and tRNAGln, and another two at the anti codon stem of tRNASer(UCN),respectively (Figure 2).

Table 5 Codon usage of the protein-coding genes of the Abisara fylloides mitogenomes

Phylogenetic analysis

At present, for the phylogenetic positions of the riodinids within papilionid butterflies, most morphological studies place them as most closely related to the lycaenids and identify the nymphalids as the closest relatives to this riodinid+lycaenid clade (de Jong et al, 1996; Ehrlich & Ehrlich, 1967; Kristensen, 1976;Scott & Wright, 1990). These relationships have been inferred using a variety of phylogenetic methods and are supported by a number of adult, larval and pupal synapomorphies. Additionally, molecular (DNA sequence of the mitochondrial NADH1 gene) or molecular plus morphological evidence also result in a monophyletic interpretation of the Riodinidae+Lycaenidae, and their sister relationship to the Nymphalidae (Wahlberg et al, 2005; Weller et al, 1996).However, based on a cladistic analysis of four foreleg characters with nine character states, Robbins (1988)suggested that the Riodinidae are more closely related to the Nymphalidae than to the Lycaenidae, and this result is supported by the nuclear 28S rRNA gene sequence data (Martin & Pashley, 1992).

There are two opinions regarding the taxonomic rank of riodinids. First, some previous studies suggest that the riodinids should be categorized into the family lycaenids as a subfamilial taxon in light of their morphological characters (Chou, 1998; de Jong et al,1996; Ehrlich, 1958; Kristensen, 1976; Scott & Wright,1990), and this opinion is supported by the molecular studies of Zou et al (2009) and Hao et al (2007). Other studies postulate that the riodinids should be classified as a separate family parallel to Lycaenidae (Harvey, 1987;Martin & Pashley, 1992; Robbins, 1988; Weller et al,1996), and this view is supported by molecular phylogenetic studies based on data from the wingless gene by Campbell et al (2000) and the combined analysis of sequences of the nuclear Ef-1a, wingless, and mitochondrial COI genes by Wahlberg et al (2005).

The ML and Bayesian trees of this study (Figure 3)showed that all the butterfly taxa in this study did not form a monophyletic unit, and a similar case was reported by Hao et al (2012). Nonetheless, both the ML and BI trees indicated that all the butterfly species were grouped into five distinct lineages: 1) the Papilionidae,including papilionids and parnassids; 2) Hesperiidae; 3)Pieridae; 4) Nymphalidae; 5) Lycaenidae+Riodinidae.The monophyly of Lycanidae + Riodinidae was strongly supported with a 100% bootstrap value in ML, and with 1.00 posterior probability value in BI. Thus, considering the results of Heikkil? et al (2012), which indicate the monophylies of lycaenids and riodinids, it is reasonable to propose that the two groups may be sisters, though the taxa sampling of riodinids in this analysis is extremely limited. Additionally, based their congruent genetic divergences compared with those between other butterfly subfamilies, the riodinids should be categorized into the Lycaenidae family as a subfamilial taxon.

Figure 2 Predicated clover-leaf secondary structures for the mitochondrial tRNA genes of Abisara fylloides

Figure 3 The Bayesian inference (BI) and maximum likelihood (ML) phylogenetic trees of main butterfly lineages based on 13 protein-coding gene sequences (Numbers on each node correspond to the posterior probability values of the BI analysis and the ML bootstrap percentage values for 1 000 replicates of ML analysis)

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