[ Note ] Int. J. Indust. Entomol. 35(1) 51-57 (2017) IJIE ISSN 1598-3579, http://dx.doi.org/10.7852/ijie.2017.35.1.51 Additional mitochondrial DNA sequences from the dragonfly, Nannophya pygmaea (Odonata: Libellulidae), which is endangered in South Korea Ah Rha Wang1,†, Min Jee Kim1,†, Sung Soo Kim2, and Iksoo Kim1,* 1College of Agriculture & Life Sciences Chonnam National University 300 Yongbong-dong, Buk-gu, Gwangju, 61186, Republic of Korea 2Research Institute for East Asian Environment and Biology, Seoul, 05207, Republic of Korea Abstract The tiny dragonfly,Nannophya pygmaea (Odonata: Libellulidae), is an endangered insect in South Korea. Previously, a partial mitochondrial DNA sequence that corresponded to a DNA barcoding region has been used to infer genetic diversity and gene flow. In this study, we additionally sequenced the barcoding region from N. pygmaea that had been collected from three previously sampled populations (40 individuals) and these sequences were combined with the preexisting data. We also selected and sequenced an additional mitochondrial gene (ND5) to find further variable gene regions in the mitochondrial genome. DNA barcoding sequences of 108 individuals from five South Korean localities showed that genetic diversity was highest in Gangjin, Jeollanam-do Province. Muuido, which was previously occupied by a single haplotype, was also found to have an identical haplotype, which confirmed the low Received : 19 Jul 2017 genetic diversity on this islet. Gene flow among populations is highly limited, and no clear Revised : 26 Jul 2017 distance- or region-based geographic partitioning was observed. Phylogenetic relationships Accepted : 26 Jul 2017 among haplotypes showed that there were no discernable haplotypes in South Korea. ND5 Keywords: provided slightly more haplotypes compared to the barcoding region in 40 individuals (14 vs. Mitochondrial DNA, 10 haplotypes in the COI gene). It also had a slightly higher within-locality diversity estimate, Genetic diversity, which suggested that ND5 had potential as mitochondrial DNA-based marker for population Nannophya pygmaea, genetic analysis. COI, © 2017 The Korean Society of Sericultural Sciences ND5, Int. J. Indust. Entomol. 35(1), 51-57 (2017) Endangered species Introduction ranges across the Indian Peninsula to Australia, including Korea (Ishida et al., 1988; Karube, 2009; Won et al., Nannophya pygmaea (Odonata: Libellulidae) is often 2009). In Korea, the species is listed as a second-degree called the “tiny” or “Scarlet Dwarf” dragonfly. It has a endangered wild animal and plant (Korean Ministry of wingspan of ~20 mm and is one of the smallest recorded Environment, 2006). Therefore, a population genetics modern odonate species (Won et al., 2009). The species analysis of N. pygmaea has been performed using a portion *Corresponding author. Iksoo Kim Department of Applied Biology, College of Agriculture & Life Sciences, Chonnam National University, Gwangju 61186, Republic of Korea. Tel: +82-62-530-5117 / FAX: +82-62-530-2079 E-mail: [email protected] †These authors contributed equally to this paper. © 2017 The Korean Society of Sericultural Sciences PB 51 Ah Rha Wang et al. Mitochondrial DNA sequences from Nannophya pygmaea of mitochondrial DNA (mtDNA) corresponding to a DNA agarose gels to confirm successful DNA amplification. The barcoding region (the 658 bp region of the COI gene). The DNA sequencing was conducted using the ABI PRISM main finding was that genetic diversity was low in Korean BigDye Terminator ver. 3.1 Cycle Sequencing Kit with N. pygmaea populations. However, the results also showed an ABI 3100 Genetic Analyzer (PE Applied Biosystems, that diversity was slightly higher in southern localities, Foster City, CA, USA). All products were sequenced from such as Gangjin and Gokseong in Jeollanam-do Province, both strands. than in other areas (Kim et al., 2007). The sequences of both strands from each individual were In this study, we additionally sampled 40 N. pygmaea from aligned using the Clustal Omega program (http://www.ebi.ac.uk/ three previously collected localities in 2016, and these data were Tools/msa/clustalo/; Sievers et al., 2011) and finalized for each combined with the pre-existing DNA barcoding sequence data to individual sample. When the sequences from each individual expand the mtDNA-based population genetic analysis data on N. differed by ≥1 nucleotide or insertion/deletion (indel) after pygmaea alignment using PAUP ver. 4.0b (Swofford, 1999), they were considered to be different haplotypes. Haplotype designations for ND5 were applied to new sequences as they were discovered (i.e., Materials and Methods NPND501, NPND502, NPND503, etc.), whereas haplotypes for the DNA barcoding region were designated by following Kim et DNA sequencing and analysis al. (2007). The mitochondrial (mt) COI gene sequences, Genetic diversity indices corresponding to a DNA barcoding region (658 bp), were amplified under the following conditions: an Within-population haplotype diversity (h) and nucleotide initial denaturation step at 94°C for 5 min, a 35-cycle diversity (π) were estimated according to Nei (1987) using amplification (94°C for 1 min, 48–52°C for 1 min, and 72°C Arlequin ver. 3.5 (Excoffier and Lischer, 2010). Maximum for 1 min), and a final extension step of 7 min at 72°C. The sequence divergence within each locality was obtained by primers for the COI sequences were designed using the extracting the within-locality estimates of the unrooted pairwise complete mt genome sequences from N. pygmaea (Jeong et distances from PAUP (Swofford, 1999). The genetic distance al., Submitted). These were NP-LCOF (5′-TTTCTACTAAT and per-generation female migration rate were estimated from CATAAGGATATTGG-3′), and NP-HCOR (5′-TAAACTTC subroutines in Arlequin ver. 3.5 (Excoffier and Lischer, 2010). CGGATGACCAAAGAATCA-3′). The variability of several Pairwise FST was used to estimate the per-generation female mt ND genes were also considered (e.g., Wan et al., 2013), migration rate, and Nm (the product of the effective population and eventually, ND5 was chosen based on amplification size, Ne, and the migration rate, m) was obtained according to efficiency, sequence divergence, and the number of variable Excoffier et al. (1992) using the equilibrium relationship: FST = sites after several individual N. pygmaea were sequenced. 1/(2Nm + 1). Any significant differences between the pairs of The primers for the amplification of a 730 bp partial localities (1,000 bootstraps) were analyzed using a permutation ND5 gene were designed using the complete mt genome test, which followed the approach described by Excoffier et al. sequences from N. pygmaea (Jeong et al., Submitted). (1992). The distances between DNA sequences were calculated These were forward, 5′-TAATAGTATATACTCCCGTG-3′, by the Kimura 2-parameters method (Kimura, 1980). In this and reverse, 5′-GCTCATGTTGAAGCTCCTG-3′. After analysis, only populations with more than two haplotypes were an initial denaturation step at 94°C for 5 min, a 30-cycle considered. This meant that four of the five populations were amplification (94°C for 1 min, 48–52°C for 1 min, and subjected to analysis. 72°C for 1 min) was conducted. The final extension step was 7 min at 72°C. The PCR product was then Phylogenetic analysis purified using a PCR Purification Kit (Qiagen, Germany). Electrophoresis was carried out in 0.5 × TAE buffer on 0.5% During the phylogenetic analysis, the GTR + GAMMA 52 53 Int. J. Indust. Entomol. Vol. 35, No. (1), pp. 51-57 (2017) + I model, which was selected by comparing the Akaike 0.680% (4 bp; data not shown). Therefore, the combined data Information Criterion scores (Akaike, 1974) using Modeltest increased the number of haplotypes, but not the maximum ver. 3.7 (Posada and Crandall, 1998), was used for the sequence divergence. maximum-likelihood (ML) method. The ML method was conducted using RAxML-HPC2 on XSEDE ver. 8.0.24 Genetic diversity indices (Stamatakis, 2006), which is found on the CIPRES Portal ver. 3.1 (Miller et al., 2010). To root the phylogenetic trees, Among the 14 haplotypes, eight haplotypes were only six N. pygmaea haplotypes, originally from Malaysia, were found in Gangjin (locality 4) and nine haplotypes were downloaded from GenBank (GenBank accession numbers found in Gokseong (locality 5). Munkyeing (locality 1) and KT991525, KT991529, KT991527, KT991526, KT991528, Suwon (locality 2) were found to contain four haplotypes, and KT991531; Low et al., 2016). The generated tree was and Muuido (locality 3) was found to contain one haplotype. viewed with FigTree software ver. 1.4.2 (tree.bio.ed.ac.uk/ BARNP10 was found in all localities at a relatively high software/figtree). frequency (data not shown). The within-locality diversity was estimated in terms of haplotype diversity (H), maximum sequence divergence (MSD), mean number of pairwise Results and Discussion differences (MPD), and nucleotide diversity (π) (Table 2). Suwon, Gangjin, and Gokseong had comparatively high Sequence analyses H values (H = 0.8154~0.8737). However, the samples collected from Muuido showed zero diversity and contained DNA sequencing of the COI DNA barcoding region only one haplotype (BARNP10; Table 2). In terms of π, and ND5 from 40 individuals identified ten haplotypes Gangjin and Gokseong had a comparatively high estimate (BARNP01, BARNP02, BARNP03, BARNP05, BARNP07, (π = 0.002465~0.002640). Although Suwon had the BARNP09, BARNP10, BARNP11, BARNP13, and second highest H, its π estimate was third behind Gangjin BARNP14) and 15 haplotypes (NPND501–NPND515) (Table and Gokseong. These diversity estimates show that, the 1), respectively. They had maximum sequence divergences populations in the southern localities, such as Gangjin and of 0.680% (4 bp) and 0.822% (6 bp), respectively (data Gokseong in Jellanamdo Province, have a relatively higher not shown). Therefore, the newly selected ND5 provided genetic diversity compared to the other localities. This result, slightly greater variability than the DNA barcoding region even after extended sampling, is consistent with previous analysis. When COI and ND5 were concatenated (1,388 bp), results obtained by Kim et al.