Sequence Divergence and Phylogenetic Investigation of the Nymphalidae (Lepidoptera: Papilionoidea) Occurring in South Korea
- Author: Wan Xinlong, Kim Min Jee, Cho Youngho, Jun Jumin, Jeong Heon Cheon, Lee Kwang Youll, Kim Iksoo
- Organization: Wan Xinlong; Kim Min Jee; Cho Youngho; Jun Jumin; Jeong Heon Cheon; Lee Kwang Youll; Kim Iksoo
- Publish: International Journal of Industrial Entomology Volume 26, Issue2, p95~112, 09 July 2013
-
ABSTRACT
As a first step toward understanding the divergence and relationships of the Nymphalidae (Lepidoptera: Papilionoidea) occurring in South Korea, cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 a (EF-1a) that comprise 3,501?3,716 bp were either sequenced (55 species) or the sequences were obtained from GenBank (23 species). The concatenated sequence divergence of six nymphalid subfamilies ranked in the following order: Danainae (10.3%), Satyrinae (9.5%), Limenitidinae (8.0%), Apaturinae (7.0%), Nymphalinae (6.7%), and Heliconiinae (6.2%). As has been reported in previous large scale international studies, the subfamilial relationships of (((((Limenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Satyrinae) + Libytheinae) + Danainae) were also confirmed, except for the switched positions between Danainae and Libytheinae, and supported all subfamilies and tribe monophylies. Unlikely consistent phylogenetic relationships among genera within the majority of tribes in Nymphalidae, a conflicting relationship within the subfamily Apaturinae was obvious, presenting
Apatura as sister to eitherMimathyma or (Mimathyma + (Sephisa + (Hestina +Sasakia ))), and both of these relationships are unconventional. Within the subfamily Limenitidinae, the genusNeptis was consistently revealed as a paraphyletic with respect to the genusAldania , requiring further taxonomic investigation of the genus. Although limited, current sequence information and phylogenetic relationships are expected to be helpful for further studies
-
KEYWORD
Sequence divergence , Mitochondrial and nuclear genes Nymphalidae , South Korea
-
About five years ago, a national project named the Korean Tree of Life (KTOL) Project was initiated in Korea, mimicking the Tree of Life (ToL). Throughout this project, many taxonomic researchers had the chance to revise, rename, and uncover by comparing the sequence-based identity to the traditional morphological data and to sequence databases such as GenBank. Furthermore, the sequence information was also utilized to complement the phylogenetic relationships of given taxonomic groups occurring in South Korea to those occurring outside the Korean peninsula, although the information is limited to the perspective of world taxonomic diversity and a large sequencebased analysis, particularly considering current trends (e.g., Mutanen
et al. , 2010; Regieret al. , 2009; Wahlberget al. , 2009).A recent ecological overview has shown ecological diversity in Korean butterflies, including Nymphalidae, in terms of biogeographic origin, distributional range, habitat characteristics, voltinism, and so on (Kim, 2012). According to Kim (2012), ~80 among ~90 species (38 genera in seven subfamilies) occurring in South Korea originated from a northern region, whereas only ~10 species, including the typical southeastern genus Parantica, originated from a tropical region. With regard to voltinism, ~60 species are univoltine, whereas the remaining species are either bivoltine, trivoltine, or multivoltine (Kim, 2012). Due mainly to such different voltinisms, differences in seasonal wing morphology, body size, and the overwintering stage are known to be prevalent.
With regard to taxonomic perspective, the Nymphalidae along with other butterflies occurring in South Korea were further welllisted by several important earlier studies since the beginning work by foreign scientists (see Kim, 2012). The subsequent majority of Nymphalidae research in South Korea has focused on introduction of individual species in illustrated books (e.g., Kim, 2002), finding and listing new species through morphological analysis (e.g., Joo
et al. , 1997; Lee and Takakura, 1981; Park, 1987), and ecological investigation of a limited number of species (e.g., Kim, 2012).For phylogenetic perspective, a report on the familial relationships of Papilionoidea was nearly a unique one that included a substantial number of species that occur in South Korea (Kim
et al. , 2010). Even so, much more research effort is required to examine taxonomic diversity in South Korea. On the other hand, in the case of world perspective on phylogeny, several studies analyzed various subgroups of Nymphalidae along with other butterflies using diverse molecular markers and morphological characters (Ehrlich, 1958; Ehrlich and Ehrlich, 1967; DeVrieset al. , 1985; Martin and Pashley, 1992; Freitas and Brown Jr, 2004). A report by Wahlberget al. (2009) is noteworthy in that it compiled a data matrix of 10 genes and 235 morphological characters with taxonomic representation of all subfamilies, tribes, and subtribes of Nymphalidae. With regard to subfamilial relationships, they found Libythinae to be the most basal lineage and placed Danainae at the next branch, presenting the other subfamilial relationships as (Satyrinae + ((Nymphalinae + Apaturinae) + (Limenitidinae + Heliconiinae))).As part of the KTOL project, in this study, 78 species of Nympahlidae were analyzed for their cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 α (EF-1α), each of which spans 3,501?3,716 bp. Twenty-three species that were previously reported by Kim
et al. (2010) and 55 species that were newly sequenced in this study together belong to 47 genera in seven subfamilies, accounting for 86% of Nympalidae occurring in South Korea (Kim, 2012). This sequence information including DNA barcode regions is expected to be helpful for species identification and for the inference of international relationships of the Nymphalidae occurring in South Korea, providing the basal data for further research. Although limited, this study contains the most diverse nymphalid species occurring in South Korea.Seventy-eight adult butterflies belonging to family Nymphalidae and representing 47 genera in seven subfamilies were used in this study (Table 1). Among them, 23 species were taken from the previous study (Kim
et al. , 2010). For phylogenetic analysis, six species of Pieridae representing three subfamilies were taken from Kimet al. (2010) for outgroups in consideration of previous studies (Ackery, 1988; Robbins, 1988; de Jonget al. , 1996; Campbellet al. , 2000). Voucher specimens were deposited in the National Institute of Biological Resources, Incheon, Republic of Korea.> DNA extraction and polymerase chain reaction (PCR)
After collection from the field, samples were frozen at -70℃ until used for molecular analyses. Total DNA was extracted with a Wizard™ Genomic DNA Purification Kit according to the manufacturer's instructions (Promega, USA).
To sequence the mitochondrial COI, 16S rRNA, and nuclear EF-1α genes, each gene was amplified into two independent fragments in most cases for easy processing. All PCR products were sequenced either directly or after cloning. The details of primer sets used for the amplification of each fragment are provided in Table 2. They were either from previous studies or newly designed. PCR amplification was conducted using AccuPower® PCR PreMix (Bioneer, Korea) under the following conditions: initial denaturation for 7 min at 94℃, followed by 35 cycles of 60 s at 94℃, 60 s at 54?58℃, and 2 min at 72℃, with a subsequent final 7-minute extension at 72℃. To confirm successful DNA amplification, electrophoresis was conducted using 0.5× TAE buffer on 0.5% agarose gel. The PCR product was subsequently purified with a PCR purification Kit (QIAGEN, Germany). Cloning was carried out using pGEM-T Easy vector (Promega, USA). The resultant plasmid DNA was isolated using a Wizard Plus SV Minipreps DNA Purification System (Promega, USA). DNA sequencing was conducted using the ABI PRISM® BigDye® Terminator v3.1 Cycle Sequencing Kit with an ABI 377 Genetic Analyzer (PE Applied Biosystems, USA). All the products were sequenced from both strands.
> Sequence analysis and phylogenetic inference
Nucleotide sequences for each gene were aligned using MAFFT ver. 6 (Katoh
et al. , 2002), with the gap opening penalty set to 1.53 and the offset value (equivalent to gap extension penalty) set to 0.5. Subsequently, the well-aligned blocks were selected using GBlocks 0.91b (Castresana, 2000), with the maximum number of contiguous non-conserved positions set to four for the COI and EF-1α genes. The 16S rRNA gene was subjected to GBlocks analysis using parameters optimized for rDNA alignments (minimum length of a block as five, allowing gaps in the half position) (Massanaet al. , 2004). Considering the general trend towards utilization of combined information including previous phylogenetic studies on Papilionoidea (Wahlberget al. , 2005, 2009; Savardet al. , 2006), phylogenetic analysis was performed using the combined three genes. Bayesian inference (BI) and Maximum likelihood (ML) methods were employed for phylogenetic reconstruction. For the BI analysis, the dataset was either unpartitioned or divided into three partitions based on each gene to assess the effect of data partitioning. The average sequence divergence (%) within a tribe, subfamily, and family for individual and combined gene sequences were estimated under an uncorrectedp -distance model using MEGA5 (Tamuraet al. , 2011).Substitution model selection was performed by comparison of Akaike Information Criterion (AIC) scores (Akaike, 1974), calculated using the Modeltest ver. 3.7 (Posada and Crandall, 1998). In each gene and combined gene sequences, the GTR (Lanave
et al. , 1984) + I + G model was selected and applied for the BI and ML methods. The ML analyses were conducted using PHYML (Guindonet al. , 2005), specifying the number of substitution rate categories as four, and the proportion of invariable site and the number of substitution sites set as the values obtained from the model test with the starting tree set as a BIONJ distance-based tree. The confidence values were evaluated via the bootstrap test with 1,000 iterations. The BI analyses were conducted using MrBayes ver. 3.1 (Huelsenbeck and Ronquist, 2001). When the partition option was employed, the model chosen by Modeltest (Posada and Crandall, 1998) was applied to each partition unlinked. Two independent runs of four incrementally heated Markov Chain Monte Carlo (MCMC) chains (one cold chain and three hot chains) were simultaneously run for one-to-three million generations depending on the dataset, with sampling conducted every 100 generations. The convergence of MCMC, which was monitored by the average standard deviation of split frequencies, reached below 0.01 within one-to-three million generations depending on the dataset, and the initial 25% of the sampled trees were discarded as burnin. The confidence values of the BI tree are presented as the Bayesian posterior probabilities (BPP).> Characteristics of sequence data
The sequence lengths of 78 nymphalid and six pierid species were 1,099 bp for COI, 1,336?1,551 bp for 16S rRNA, and 1,066 bp for EF-1α (Table 1). Both COI and EF-1α did not have any insertions/deletions. The concatenated sequences of the three genes ranged 3,501?3,716 bp among species. However, the conserved blocks selected by GBlocks analysis (Castresana, 2000) eventually gave a total of 3,404 bp, composed of 1,243 bp from 16S rRNA (64% of original sequences including 92 indels),
1,097 bp from COI (99% of original sequences), and 1,064 bp from EF-1α (99% of original sequences).
The average A/T content of COI, 16S rRNA, and EF-1α were 70.6%, 84.2%, and 49.2%, respectively (Table 1). This result provided evidence of the A/T bias in mitochondrial genes, but not for nuclear genes. Between the two mitochondrial genes, the 16S rRNA gene showed much higher A/T content than that of COI gene. The A/T content of protein coding genes and 16S rRNA in Lepidoptera was approximately 75.7%?81.5% and 81.4%?85.1%, respectively (Kim
et al. , 2011), indicating somewhat higher A/T content in 16S rRNA.The average sequence divergence of within-subfamily ranged from 7.8%?14.5% in COI, 5.1%?9.3% in 16S rRNA, and 3.2%?9.5% in EF-1α, with the concatenated divergence ranging from 4.7%?10.3% (Table 3). In six nymphalid subfamilies, the concatenated divergence ranked in the following order: Danainae (10.3%), Satyrinae (9.5%), Limenitidinae (8.0%), Apaturinae (7.0%), Nymphalinae (6.7%), and Heliconiinae (6.2%). The same rank of sequence divergence was also found in the individual COI and 16S rRNA genes, but that for the EF-1α gene ranked in the order of Satyrinae, Danainae, Limenitidinae, Nymphalinae, Apaturinae, and Heliconiinae (Table 3), indicating different modes of evolution between the two types of genes.
> Subfamilial relationships in Nymphalidae
All the analyses based on concatenation of the COI gene plus the EF-1α and 16S rRNA genes, regardless of analytical methods and partitioning strategies, concordantly recovered all nymphalid subfamilies and tribes as monophyletic groups with high nodal supports (Fig. 1). This result was consistent with the latest phylogenetic study using combined molecular and morphological data (Wahlberg
et al. , 2009) and early morphological studies (Ehrlich, 1958; Ehrlich and Ehrlich, 1967).With regard to subfamilial relationships, all analyses, regardless of analytical methods, supported Danainae as the most basal lineage of Nymphalidae, placing it as the sister to Libytheinae and the remaining subfamilies with high nodal supports (1.00 by BI and 100% by ML analyses) (Fig. 1). This result has never been proposed in previous studies, except for that by Kim
et al. (2010), wherein the concatenated COI, 16S rRNA, and EF-1α genes were also used for phylogenetic reconstruction among true butterfly families. Several phylogenetic studies based on morphological (Ehrlich and Ehrlich, 1967; Freitas and Brown Jr, 2004) and/or molecular data (Wahlberget al. , 2003, 2009) placed Danainae as the sister to the remaining subfamilies of Nymphalidae, but placed Libytheinae as the most basal lineage. Such placement has been widely supported by morphological characteristics, such as lack of reduced female foreleg, an apomorphic feature found in all other Nymphalidae (Ehrlich and Ehrlich, 1967; Scott, 1984; Freitas and Brown Jr, 2004) and/or molecular data (Martin and Pashley, 1992; Wahlberget al. , 2009). Furthermore, host plant and geographic distribution also suggested that this subfamily was an outgroup of the remaining Nymphalidae, as the most basal subfamily (Ackery, 1984; Freitas, 1999; Vane-Wright, 2003). Thus, the placement of Danainae at the most basal branch, instead of Libytheinae in our study, is likely a consequence of limited sampling.The subfamily Satyrinae was consistently placed as the next basal lineage after Libytheinae and Danainae in all analyses with moderate and low supports by BI (0.73 and 0.75) and by ML analyses (55%), respectively (Fig. 1), providing the subfamilial
relationships (((Limenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Satyrinae). This relationship is concordant with that proposed by Wahlberg
et al. (2009), but not to those obtained by several earlier morphology-based studies (Ehrlich, 1958; DeVrieset al. , 1985; Freitas and Brown, 2004).The sister relationships between Nymphalinae and Apaturinae were strongly supported in the present study (1.00 by BI and 100% by ML analyses). This result was consistent with that revealed by Ehrlich and Ehrlich (1967) and Wahlberg
et al. (2009), but differed from that by Martin and Pashley (1992), wherein 28S and 18S rRNA data were used and were consistent with the results demonstrated by Freitas and Brown Jr (2004), wherein morphological characters from all stages, such as the color of eggs, caudae of larvae, and wing patterns of adults were used. Martin and Pashley (1992) demonstrated neither Apaturinae nor Nymphalinae to be monophyletic. In the case of Apaturinae, one genus (Doxocopa ) was placed as the sister to Danainae, leaving another genus (Asterocampa ) as the sister to this group. In the case of Nymphalinae, one genus (Vanessa ) was placed as the sister to the group composed of ((Danainae +Doxocopa ) +Asterocampa ). Freitas and Brown (2004) proposed Apturinae as a monophyletic group, but placed it as the sister to Satyrinae, leaving them as the most recently derived lineages of Nymphalidae. However, Nymphalinae was also recovered as a nonmonophyletic group, placing the majority of Nymphalinae (Nymphalini, Kallimini, and Melitaeini) as sisters to Heliconiinae, while the single tribe Coeini was placed as the sister to Limenitidinae. The sister relationship between Limenitidinae and Heliconiinae was supported with the highest nodal supports (1.00 by BI and 100% by ML analyses), consistent with that revealed by Wahlberget al. (2009).Overall, the phylogenetic analyses in the present study yielded each subfamily and tribe as a monophyletic group, and the subfamilial relationships within Nymphalidae as (((((Limenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Satyrinae) + Libytheinae) + Danainae).
> Internal relationships within tribes and subfamilies
In the tribe Satyrini of Satyrinae, six of 14 genera (
Oeneis ,Melanargia ,Ypthima ,Lethe ,Mycalesis , andCoenonympha ) represented by more than one species were all consistently shown to be monophyletic groups in all analyses (Fig. 1). Concordantly in all analyses,Coenonympha (three species) was placed as the most basal lineage of Satyrini with high nodal supports (1.00 by BI and 100% by ML analyses), consistent with that reported by Martinet al. (2000), wherein 16S rRNA was used. The remaining genera were subdivided into two groups, formingYpthima as the sister to the group composed of (((((Oeneis +Minois ) +Eumenis ) +Melanargia ) +Aphantopus ) +Erebia ) on one hand (1.00 by BI and 51% by ML analyses), and the group composed of ((Kirinia +Lasiommata ) +Pararge ) as the sister to (Mycalesis + (Lethe +Ninguta )) on the other hand (1.00 by BI and 68% by ML analyses) (Fig. 1). This result conflicted with previous studies. For example, Martinet al. (2000) reported the genusLasiommata at the most basal branch and the genusPararge at next branch, placing the genusCoenonympha as the sister to the remaining Satyrini based on concatenated mitochondrial 16S rRNA and ND1 genes. Penaet al. (2011) using substantially large molecular data reported that the group (((Mycalesis +Lethe ) +Pararge ) +Coenonympha ) was basal to the Satyrini. Probably, an expended sampling and sequences for the Satyrini might be needed to better understand the relationships within the Satyrini that occur in South Korea.The genera
Apatura ,Mimathyma (=Athymodes ), and Hestina, that were sampled for more than one species in Apaturinae were all consistently revealed as monophyletic groups with high nodal supports (1.00 by BI and 100% by ML analyses) (Fig. 1). All analyses consistently revealedDravira as the most basal lineage of Apaturinae, forming the remaining genera as a monophyletic group with high nodal supports (1.00 by BI and 100% by ML analyses), consistent with previous studies (Ohshimaet al. , 2010). However, the internal relationship within the remaining genera fluctuated depending on the analytical method. BI analyses, regardless of data partitioning, showed the group composed of (Apatura +Mimathyma ) as the sister to the group composed of (Sephisa + (Hestina +Sasakia )), whereas the ML analysis supportedApatura as the sister to (Mimathyma + (Sephisa + (Hestina +Sasakia ))), although the nodal supports for the latter grouping were obviously lower (Fig. 1). Nevertheless, both relationships conflicted with previous studies, which either suggestedApatura as the sister to Hestina + Sasakia on the basis of a single mitochondrial gene (Zhanget al. , 2008) orSephisa as the sister toApatura +Mimathyma on the basis of combined mitochondrial and nuclear genes (Ohshimaet al. , 2010). The Apaturinae is one of the unresolved nymphalid groups, consisting of 430 described species belonging to 20 genera. However, the taxon-specific morphologies, such as an extremely elongated phallus and saccus in male genitalia that are absent from other butterfly species, support the monophyly of Apaturinae (Le Moult, 1950; Shirozu and Saigusa, 1971; Ackeryet al. , 1999). As has been supported by molecular data (Wahlberget al. , 2003; Zhanget al. , 2008; Ohshimaet al. , 2010) and combined molecular and morphological data (Wahlberget al. , 2005, 2009), the monophyly of this subfamily is also supported in our study (Fig. 1).In the subfamily Nymphalinae, the tribe Nymphalini (seven genera) and the tribe Melitaeini (three genera) each were well recovered as monophyletic groups with strong support in all analyses (Fig. 1). Two genera,
Polygonia andVanessa , represented by more than one species in the Nymphalini, were consistently recovered as monophyletic groups in all analyses. Furthermore, all analyses concordantly supported the phylogenetic relationships ((((((Nymphalis +Kaniska ) +Polygonia )) +Inachis ) +Vanessa ) +Araschnia ) within Nymphalini and ((Melitaea +Mellicta ) +Euphydryas ) within Melitaeini (Fig. 1). Placed as the sister toVanessa , the monophyly of theNymphalis group (Nymphalis ,Kaniska ,Polygonia , andInachis ) within Nymphalini was also recovered in our study with high nodal supports (1.00 by BI and 100% by ML analyses), as has been supported in previous studies (Wahlberg and Zimmermann, 2000; Nylinet al. , 2001; Wahlberget al. , 2005). The positions ofAraschnia andEuphydryas each as the most basal lineage of Nymphlini and Melitaeini, respectively, were consistent with previous studies (Wahlberg and Zimmermann, 2000; Nylinet al. , 2001; Wahlberget al. , 2005).Nymphalinae is a well-supported monophyletic group that includes the four well-defined tribes: Nymphalini, Coeini, Melitaeini, and Kallimini (Freitas and Brown Jr, 2004; Wahlberg
et al. , 2003, 2005). In Melitaeini, both Wahlberg and Zimmermann (2000) using mtDNA sequences (COI and 16S rRNA), and Wahlberget al. (2005) using combined mitochondrial and nuclear sequences, placedEuphydryas as the most basal lineage of Melitaeini, consistent with the present study. Nylinet al. (2001), Wahlberget al. (2003), and Wahlberget al. (2005) established the monophyly of the Nymphalini and the close relationships amongKaniska ,Nymphalis , andPolygonia , as was verified in this study. However, the relationships of other genera within Nymphalini differ from the current study, possibly due to limited sampling in this study.With respect to the tribe Argynnini in Heliconiinae, Simonsen (2006) subdivided this tribe into three subtribes based on 141 characters derived from wing and genitalia of males and females, with Euptoietina consisting of the genus
Euptoieta , Yrameina consisting of the generaYramea andBoloria , and Argynnina consisting of the generaProkuekkenthaliella ,Issoria ,Brenthis , andArgynnis . By combining the morphological characters with new molecular data, Simonsenet al. (2006) subsequently suggested that the tribe Argynnini should be subdivided into four subtribes, placing the genusBoloria in a new subtribe Boloriina and proposed the subtribal relationships (Euptoietina + (Yrameina + (Boloriina + Argynnina))). In this regard, all genera employed in our study, except for Brenthis and Clossiana, belong to the subgenera of the genusArgynnis , whileClossiana belongs to a subgenus of the genusBoloria . Consistent with the data of Simonsen (2006) and Simonsenet al. (2006), our results indicated the three genera within the tribe Argynnini monophyletic groups and internal relationships among genera as ((Argynnis +Brenthis ) +Boloria ) in all analyses. Simonsen’s genus Argynnis was consistently split into two clades with high supports in all analyses in our study: the first one comprising one subgeneraFabriciana (represented byF. nerippe andF. adippe ) and the other composed of the remaining Argynnis including subgeneraArgynnis ,Nephargynnis ,Damora ,Argyronome ,Childrena , andArgyreus (Fig. 1). This result is also consistent with the data of Simonsenet al. (2006).Within the subfamily Limenitidinae, the tribe Neptini composed of two genera (
Neptis andAldania ) was recovered as a monophyletic group. Nevertheless, the genusNeptis represented by 10 species was revealed as paraphyletic with respect to the genusAldania in all analyses (Fig. 1).Aldania raddei represented for the genusAldania is somewhat larger in adult body size, although male genitalia characters are typical (Kim, personal communication). Nevertheless, the genusAldania is known as an offshoot ofNeptis from eastern Russia and China in temperate Palearctic region (Tuzovet al. , 1997) and even has been considered synonymous to the genusNeptis in several studies (Yuan and Wang, 1994; Huang, 2003). Thus, taxonomic revision for the genus might be required after further critical analysis.The tribe Limenitidini was sampled only for one genus of
Limenitis (eight species) in our study. The monophyly ofLimenitis was strongly supported in BI analyses (1.00), while moderately supported in ML analysis (79%) (Fig. 1). In contrast to our study, using the mitochondrial CytB gene, Wuet al. (2007) recovered asLimenitis paraphyletic. Zhanget al. (2011) also revealedLimenitis as paraphyletic by the ML method based on the COI gene alone. Although Zhanget al. (2011) reported a monophyleticLimenitis by the maximum parsimony method, the bootstrap value was very low (9%).Overall, stable phylogenetic relationships among genera within tribes or subfamilies of Nymphalidae were obtained in all analyses, except for the conflict within Apaturinae that
Apatura was placed as the sister to eitherMimathyma or (Mimathyma + (Sephisa + (Hestina +Sasakia ))). Nevertheless, the current study is the first extensive study for the Nymphalidae occurring in South Korea. Thus, the limitations of taxon sampling and sequence quantity were unavoidable, resulting in far less for completion compared with international criteria. Nevertheless, we expect the current work might be informative as the basal data for future work on the Nymphalidae occurring in South Korea.-
21. Kim MJ, Kang AR, Jeong HC, Kim KG, Kim I (2011) Reconstructing intraordinal relationships in Lepidoptera using mitochondrial genome data with the description of two newly sequenced lycaenids, Spindasis takanonis and Protantigius superans (Lepidoptera: Lycaenidae). [Mol Phylogenet Evol] Vol.61 P.436-445
-
34. Ohshima I, Tanikawa-Dodo Y, Saigusa T, Nishiyama T, Kitani M, Hasebe M, Mohri H (2010) Phylogeny, biogeography, and hostplant association in the subfamily Apaturinae (Insecta: Lepidoptera: Nymphalidae) inferred from eight nuclear and seven mitochondrial genes. [Mol Phylogenet Evol] Vol.57 P.1026-1036
-
38. Regier JC, Zwick A, Cummings MP, Kawahara AY, Cho S, Weller S, Roe A, Baixeras J, Brown JW, Parr C, Davis DR, Epstein M, Hallwachs Hausmann A, Janzen DH, Kitching IJ, Solis MA, Yen S-H, Bazinet al, Mitter C (2009) Toward reconstructing the evolution of advanced moths and butterflies (Lepidoptera: Ditrysia): an initial molecular study. [BMC Evol Biol] Vol.9 P.280
-
[Table 1.] Full taxon names, nucleotide frequencies, gene sizes, and GenBank accession numbers for the species included in this study
-
[Table 2.] Primers used for the amplification of COI, 16S rRNA, and EF-1α genes
-
[Table 3.] Average sequence divergence within tribe, subfamily, and family (%)
-
[Fig. 1.] Phylogenetic analyses using the concatenated mitochondrial COI, 16S rRNA, and nuclear EF-1α genes. Six species of Pieridae were used as outgroups. (A) Bayesian inference phylogeny obtained without data partitioning. The numbers at each node specify Bayesian posterior probabilities (BPP). (B) Maximum likelihood. The numbers at each node specify bootstrap percentage of 1000 pseudoreplicates. (C) Bayesian inference phylogeny obtained with three partitions based on each gene. The numbers at each node specify BPP.