Journal of Asia-Pacific Biodiversity 8 (2015) 287e294

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Journal of Asia-Pacific Biodiversity

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Original article Molecular comparison of the Junonia (: ) in Myanmar

Nan Zarchi Win a, Eun Young Choi a, Deok-Jin Jang a, Jinyoung Park b, Jong Kyun Park a,* a Department of Applied Biology, College of Ecology and Environmental Sciences, Kyungpook National University, Sangju, Republic of Korea b Department of Nature Survey, National Institute of Ecology, Seocheon, Republic of Korea article info abstract

Article history: Molecular comparison of belonging to the genus Junonia collected from Myanmar was completed Received 22 September 2015 using mtDNA sequence data from 605-bp cytochrome oxidase subunit I (COI). Six species were Received in revised form sequenced, aligned, and used to construct phylogenetic trees. The base composition of the COI sequences 9 October 2015 was 37.8% T, 15.4% C, 31.4% A, and 15.4% G, revealing strong AT bias (69.2%). The sequence distance of Accepted 12 October 2015 Junonia ranged from 1.5% to 9.0%. Nucleotide substitution primarily occurred through transition rather Available online 19 October 2015 than transversion. Phylogenetic trees were constructed by the neighbor-joining (NJ) and maximum likelihood methods, using Hypolimnas misippus as the outgroup. Both trees had almost identical topol- Keywords: COI gene ogies. All COI sequences of each species fell in the same cluster as those of the same species obtained Junonia from the National Center for Biotechnology Information (NCBI). Species in Junonia exhibited the molecular phylogeny following relationships: (((J. orithya þ J. hierta) þ J. lemonias lemonias) þ J. almana almana) þ (J. atlites þ Myanmar J. iphita). The clustering results were almost identical to current morphological classification. Copyright Ó 2015, National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). Production and hosting by Elsevier. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction short tail, with this characteristic also being observed in the anal angle. In contrast, the hind wing is nearly evenly rounded in other The butterfly genus Junonia Hübner, 1816, is an important part of species. The larvae are rather stout, and are of almost of equal the Nymphalidae family (subfamily Nymphalinae). The butterflies thickness along the entire body, which is armed with strong, from this genus are commonly known as Buckeyes and Pansies. The branched spines. The larvae feed on a wide variety of plants (De genus contains 33 species that are distributed throughout all major Niceville 1886). biogeographical regions of the world, except the Palaearctic The extant members of this genus are predominantly tropical, (Kodandaramaiah and Wahlberg 2007). The adults of these species with 29 species being confined to tropical latitudes. One, four, 17 (of are medium to large sized and are good fliers. The various members which 15 are endemic), 10 (of which 8 are endemic), and three of the genus differ markedly in color, but have several character- species are distributed in the Nearctic region (North America), the istics in common, including prominent maculae in the discal cell of Neotropics, the Afrotropical region, the Oriental region, and the the forewing and circular ocelli (or eyespots) on the hind wings Australasian region, respectively (Kodandaramaiah and Wahlberg (Kodandaramaiah 2009). The eyespots on the wings are orange, 2007). Of the 10 species from the Oriental region, six have been blue, or pink, and are sometimes large. Eyespot characteristics (e.g., recorded in Myanmar (Bingham 1905; Kinyon 2004). The genera number, arrangement, size, and coloration) markedly vary across Junonia and Hübner have often been treated as congeneric species, despite being conserved within species. In some species, (Hemming 1934). However, Lesse (1952) showed that Junonia and the third median vein on the outer margin of the hind wing forms a Precis are quite distinct based on male genitalia and hind wing characteristics. Moreover, recent molecular studies have confirmed that they are distinct genera, and are not even sister genera (Wahlberg et al 2005b). The genus Junonia is well known, with the * þ þ Corresponding author. Tel.: 82 42 530 1215; fax: 82 42 530 1218. species being used as model organisms for evolutionary studies, E-mail address: [email protected] (J.K. Park). using the development of eyespots and color patterns, and for Peer review under responsibility of National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). studies on the chemical ecology and evolution of host plant http://dx.doi.org/10.1016/j.japb.2015.10.003 pISSN2287-884X eISSN2287-9544/Copyright Ó 2015, National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). Production and hosting by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 288 NZ Win et al. / Journal of Asia-Pacific Biodiversity 8 (2015) 287e294 preferences (Bowers 1984; Kodandaramaiah 2009; Monteiro and Table 1. Basic statistics of the Junonia sequences. Prudic 2010; Nijhout 1980). Position No. of No. of No. Empirical base The classification of closely related lepidopteran species based sites variable informative frequencies (%) on morphological features alone presents several difficulties and the risk of inaccuracy because the function of certain attributes TCAG differs in different environments, leading to the prevalence of All positions 605 75 39 37.8 15.4 31.4 15.4 several biotypes (Linares et al 2009). Recent molecular marker First position 202 13 8 27 13.9 31.0 18.1 Second position 202 1 e 43 25.8 14.9 16 techniques have facilitated the assessment of genetic diversity, Third position 201 61 31 43 6.5 48.4 1.9 improving the accuracy of genotyping, classification, inventorying, and molecular phylogenetic studies (Silva et al 2010). Mitochon- drial and nuclear genes, such as elongation factor-1a gene and Table 2. The number and pattern of nucleotide substitution in the Junonia sequences wingless gene, have been widely used for the molecular study of (from row to column). butterflies (Caterino et al 2000; Kandul et al 2004; Monteiro and AT C G Pierce 2001; Wahlberg et al 2003, 2005a; Zakharov et al 2004a). DNA sequences of mitochondrial genes have been recently used to A e 6.84 2.79 2.95 e infer species-level phylogenies, due to the ease of polymerase T 5.7 15.87 2.78 e fi C 5.7 38.94 2.78 chain reaction (PCR) ampli cation and due to maternal inheri- G 6.06 6.84 2.79 e tance, in addition to the lack of recombination and relatively high mutation rates. Several studies have used mtDNA sequences to investigate the phylogenetic relationships of certain groups of 0.046 butterflies (Brower 1997; Brunton and Hurst 1998; Kankare and Shaw 2004; Monteiro and Pierce 2001; Murray and Prowell 0.039 2005; Zimmermann et al 2000). Of the identified mitochondrial genes, the cytochrome c oxidase subunit I (COI) region has been 0.031 Transition used to identify various (Hebert et al 2003a, 2003b, Transversion 0.023 2004b; Ward et al 2005). In the eastern USA and northwestern

Costa Rica, DNA barcodes for the COI region have been shown to be 0.015 highly reliable for species-level identifications within the Lepi- doptera, with a success rate of >97% for w2000 morphologically 0.008 defined taxa (Hajibabaei et al 2006; Hebert et al 2003a, 2010; of transitions/transversions Proportion 0.000 Janzen et al 2005). COI sequence divergence of 3.6% occurs 0.0134 0.0256 0.0378 0.0500 0.0622 0.0744 0.0866 within lepidopteran species complexes (Lee et al 2005; Sperling Pairwise distance et al 1996; Sperling and Hickey 1994), and may even exceed 5% in some cases (Hebert et al 2004a). Figure 1. The number of transitions and transversions plotted against the uncorrected Myanmar is the second largest country in Southeast Asia. It is pairwise sequence divergence. highly biodiverse, with a rich variety of habitat types largely arising from its unusual ecological diversity. Talbot (1939) and Kinyon Table 3. Percentage pairwise distances among six Junonia species. Intraspecific (2004) recorded 1014 and 1250 species of butterflies in Myanmar, divergence is shown in parenthesis. respectively. Wikramanayake et al (2002) reported northern Myanmar to be one of the richest locations worldwide for Lepi- J. lemonias J. almana J. orithya J. atlites J. iphita J. hierta lemonias (0) almana (0) (0.3) (0.08) (0.3) (1.04) doptera. Nevertheless, reports on the systematics, morphology, and molecular phylogeny of Myanmar butterflies remain limited. The J. lemonias e current work represents the first comprehensive molecular analysis lemonias J. almana 6.7 fl fi of Buckeye butter ies (Junonia) in Myanmar. Speci cally, we aimed almana to establish the COI sequence of Junonia butterflies from Myanmar J. orithya 3.1 5.7 for taxonomic identification, and to investigate the phylogenetic J. atlites 8.4 7.5 8.1 relationship among the species within this genus. J. iphita 7.3 7.3 7.5 2.4 J. hierta 4.8 6.9 1.5 9.0 8.8 e

Materials and methods J. ¼ Junonia.

Sample collection and DNA extraction mixture was pipetted into the DNeasy mini spin column, which was Six species from the genus Junonia were used to analyze in this then placed in a 2 mL collection tube and centrifuged at 8000 rpm study, in addition to Hypolimnas misippus, which was used as the for 1 minute. The mini spin column was transferred to a new 2 mL outgroup. All samples were collected from Popa Mountain (20 530 collection tube. An amount of 500 mL of buffer AW1 was then added N and 95 150 E), Myanmar, and were preserved by dehydration in and centrifuged for 1 minute at 8000 rpm. The mini spin column small envelopes. Genomic DNA was extracted from two legs of the was then transferred to a new 2 mL collection tube again, and dried butterflies, according to the protocol of the DNeasy blood and 500 mL buffer AW2 was added. The mixture was then centrifuged tissue kit (Qiagen, Valencia, CA, USA). The legs were dissected and for 3 minutes at 14,000 rpm. Subsequently, the spin column was left placed in a 1.5 mL microcentrifuge tube and 180 mL buffer ATL and at room temperature for a few minutes to dry. The mini spin col- 20 mL protein K solution were added before vortexing for a few umn was transferred to a clean 1.5 mL microcentrifuge tube, and seconds. The mixture was then incubated at 56C overnight. Sub- 15 mL buffer AE was directly added on to the DNeasy membrane, sequently, 200 mL buffer AL was added and vortexed to ensure the and was incubated at room temperature for 10 minutes, after which solution was thoroughly mixed, after which 200 mL 100% ethanol 15 mL buffer AE was added again. After centrifuging for 3 minutes at was added and the solution was again vortexed. The homogeneous 13,000 rpm to elute, the resulting DNA was maintained at 20C. The Figure 2. Alignment of partial cytochrome oxidase subunit I (COI) sequences in six species of the genus Junonia. Identity with the first sequence is indicated by dots. 290 NZ Win et al. / Journal of Asia-Pacific Biodiversity 8 (2015) 287e294

Figure 3. Phylogenetic tree constructed using neighbor-joining (NJ) method with cytochrome oxidase subunit I (COI) sequences in species of the genus Junonia. Hypolimnas misippus was used as an outgroup. The examined species of Junonia is indicated by bold font. Scientific names of host species are shown at the terminal nodes and GenBank accession numbers are in parentheses. The numbers above the branches indicate bootstrap probabilities (percent). The bar represents phylogenetic distance. concentrations of the purified DNA samples were determined by pairwise distances were estimated using the Kimura 2-parameter. using a nanophotometer spectrophotometer. The number of transitions and tranversions were plotted against sequence divergence values to determine substitution saturation PCR amplification and DNA sequencing using the program DAMBE (Xia 2013). Phylogenetic analyses were constructed using MEGA 6 with neighbor-joining (NJ) (Saitou and The COI sequence was amplified using LCO (50-GGTCAA- Nei 1987) and maximum likelihood methods (Pond et al 2005). CAAATCATAAAGAT ATTGG-30) and HCO (50-TAAACTTCAGGGTGAC- The NJ analyses were conducted using the Kimura 2-parameter CAAAAAATCA-30)(Folmer et al 1994). PCR was performed in a total distance estimate, with 1000 bootstrap replications. The COI gene volume of 20 mL. The cycle conditions were as follows: denaturation sequences in the phylogenetic tree, obtained from the Myanmar at 94C for 7 minutes; 35 cycles each at 94C for 1 minute, 54C for butterfly samples, were compared with previously described 1 minute, and 72C for 2 minutes; and a final extension at 72C for 7 Junonia retrieved from the GenBank. minutes. The PCR products were separated by using 1% agarose gel electrophoresis, and purified using a Wizard kit. Results and discussion

Phylogenetic analysis The fragment length of the COI sequences of the six Junonia species was 605 bp. The nucleotide sequences have been deposited The purification products were sent to Macrogen (Seoul, Korea) in the National Center for Biotechnology Information (NCBI) data- for sequencing. The sequences were edited using the Editseq pro- base under the accession numbers KP979792, KP997222, gram (DNASTAR, 6). The sequences were aligned against the pub- KP997223, KP997224, KP997225, and KP997226. The sequences lished reference sequences in the GenBank using the BLAST were compared against those species previously deposited in the program (Altschul et al 1997; Schäffer et al 2001). NCBI database, and showed 99e100% similarity. Multiple sequence alignments were performed using the Clustal Within the examined 605 bp, there were 75 variable sites and 39 W multiple alignments function in BioEdit version 7.0 (Hall 1999). parsimony informative sites (Table 1). Like other protein coding Nucleotide composition and patterns of nucleotide substitution genes, most variations occurred at the third codon position. Among were calculated by the MEGA 6 program (Tamura et al 2013), and informative sites, eight were in the first position and 38 were in the NZ Win et al. / Journal of Asia-Pacific Biodiversity 8 (2015) 287e294 291 third position. The mean base composition of the COI sequences of divergence. This result showed that substitutions did not become was 37.8% T, 15.4% C, 31.4% A, and 15.4% G. There was a strong AT saturated in either transitions or transversions, and so could be bias (69.2%), which is in accordance with previous publications on used for further phylogenetic analyses. mitochondrial genomes (DeSalle et al 1987; Simon et al The pairwise distance was calculated by the MEGA 6 program. 1994). The AþT content of the third, second, and first codon posi- The intraspecific nucleotide divergence for each species was tions of the COI fragment was 91.4%, 57.9%, and 58%. The high AT calculated using the sequences of the same species from other percentage at the third codon position might be caused by a change countries (Thailand, India, Pakistan, China, Taiwan, Japan, and in the mutation pressure from GC towards AT. This pressure Sweden) deposited in the NCBI database. Intraspecific divergences imposed strong codon usage bias, favoring A-ending and T-ending ranged from 0.00% to 1.03% with a mean of 0.3% (Table 3). A min- codons, resulting in a distinct mutation-selection balance for genes imum intraspecific nucleotide divergence of 0.00% was obtained for encoded on opposite strands (DeSalle et al 1987; Simon et al 1994). lemonias and , while a maximum Two types of nucleotide substitution were observed: transition intraspecific nucleotide divergence of 1.03% was found with Junonia and transversion. Transition was more common than transversion hierta. Widely distributed species exhibited high intraspecific (Table 2 and Figure 1), supporting the general rule. Ebersberger et al divergence. The 0.00% sequence divergence showed that the DNA (2002) suggested transitions occur more frequently in genomes, sequences overlapped, possibly due to low variation among in- and might occur if disfavored tautomeric forms of the four bases are dividuals of the same species from different geographical locations. misincorporated during DNA replication to form AeC and GeT In contrast, the maximum intraspecific nucleotide divergence of transition mispairs (Watson and Crick 1953). In addition, the 1.03% indicated that there is a higher degree of variation among transition between T and C was higher than the transition between individuals of the same species from different locations. A and G in the current study. The highest transversion occurred Muhammad et al (2013) reported that there is a weak relationship from A to T, and G and T in all four types of transversion (Table 2). between geographical extent of a species and its intraspecific The program DAMBE was used to plot nucleotide substitutions divergence. Similarly, Lukhtanov et al (2009) concluded that against sequence divergence values (Figure 1). The number of geographical distance is often associated with an increased genetic transitions and transversions increased with pairwise divergence. divergence, but that the increase is too small to impede the iden- More transitions than transversions occurred at almost every level tification of species. The interspecific nucleotide divergence among

Figure 4. Phylogenetic tree constructed using maximum likelihood (ML) method with cytochrome oxidase subunit I (COI) sequences in species of the genus Junonia. Hypolimnas misippus was used as an outgroup. The examined species of Junonia is indicated by bold font. Scientific names of host species are shown at the terminal nodes and GenBank accession numbers are in parentheses. The numbers above the branches indicate bootstrap probabilities (percent). The bar represents phylogenetic distance. 292 NZ Win et al. / Journal of Asia-Pacific Biodiversity 8 (2015) 287e294

Figure 5. Junonia species collected from Myanmar. A, J. hierta;B,J. orithya;C,J. lemonias lemonias;D,J. almana almana;E,J. atlites;F,J. iphita. the six species ranged from 1.5% to 9.0%, with an average of 6.3% cluster as those of the same species obtained from the NCBI. (Table 3). The highest distance of 9.0% was obtained between H. misippus was used as the outgroup. The Junonia genus was a and J. hierta. The shortest distance of 1.5% was ob- monophyletic group in all phylogenetic trees, which was consistent tained between and J. hierta. Although sequence with Zhang et al (2008) and Kodandaramaiah and Wahlberg divergence at COI of >2% is used for species discrimination in (2007). All species, except J. orithya, were strongly supported lepidopterans (Hebert et al 2003b), low interspecific divergence monophyletic groups in this study. Kodandaramaiah and Wahlberg may be observed due to the presence of interspecies hybridization, (2007) also reported that the widespread species, J. orithya, was a which is a well-known phenomenon in many butterflies. Sequence polyphyletic unit. J. orithya was closely related to J. hierta, and divergences ranging from 0% to 1.2% have been found within many formed a monophyletic group with strong support. According to species of Papilio that are widely distributed across Africa and the morphological characteristics, De Niceville (1886) stated that Madagascar (Zakharov et al 2004b). Moreover, the gap between J. hierta and J. orithya should be in the same group because the maximum intraspecific and minimum interspecific distances has following characteristics are the same: the outline of the forewing been used for species delimitation in various groups (Hebert is less falcate, the hind wing is evenly rounded, the margin is less et al 2004b; Meier et al 2006, 2008; Meyer and Paulay 2005; sinuated in both wings, and the anal angle of the hind wing does Puillandre et al 2012). Figure 2 presents the alignment of the COI not form a tail. These two species were then clustered with sequences for the genus Junonia in Myanmar, presenting both J. lemonias lemonias, with strong support in all trees. Then J. almana variation and similarities. Variation in the nucleotide sequence is a almana was a sister group to the clade of J. lemonias lemonias þ fundamental property of all living organisms, and may be used for (J. orithya þ J. hierta) in all trees. The position of as the their identification and phylogenetic status. sister group to J. atlites was strongly supported in all trees. The six Molecular phylogenetic trees were constructed for the COI gene Junonia species have the following relationships: (((J. orithya þ using the NJ, and maximum likelihood methods. The trees showed J. hierta) þ J. lemonias lemonias) þ J. almana almana) þ (J. atlites þ almost identical topologies (Figures 3 and 4). Almost all COI se- J. iphita). These results are accordance with the findings of quences of each species fell with high bootstrap values in the same Wahlberg et al (2005b). Similarly, Zhang et al (2008) reported a NZ Win et al. / Journal of Asia-Pacific Biodiversity 8 (2015) 287e294 293 stable relationship among Junonia species in China, whereby: Kodandaramaiah U, Wahlberg N. 2007. 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