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 either Mimathyma or (Mimathyma + (Sephisa + (Hestina + Sasakia ))), and both of these relationships are unconventional. Within the subfamily Limenitidinae, the genus Neptis was consistently revealed as a paraphyletic with respect to the genus Aldania, requiring further taxonomic investigation of the genus. Although limited, current sequence information and phylogenetic relationships are expected to be helpful for further studies
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
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
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
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
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
>
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).
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
Primers used for the amplification of COI, 16S rRNA, and EF-1α genes
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
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
>
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),
[Table 3.] Average sequence divergence within tribe, subfamily, and family (%)
Average sequence divergence within tribe, subfamily, and family (%)
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
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
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
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
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
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 (
The genera
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,
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
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
Within the subfamily Limenitidinae, the tribe Neptini composed of two genera (
The tribe Limenitidini was sampled only for one genus of
Overall, stable phylogenetic relationships among genera within tribes or subfamilies of Nymphalidae were obtained in all analyses, except for the conflict within Apaturinae that