Rikunomizu（Limnology in Tokai Region of Japan）74 : 53 - 56（2016）

ReportMolecular phylogenetic analysis of Nannophya pygmaea from the Tono areaof Gifu Prefecture, Japan, and South Korea
Motoyasu MINAMI1), Yui AJIOKA2) and Sayaka NAKAMURA1)
Abstract
To investigate the phylogeny of the Tiny dragonfly, Nannophya pygmaea (Odonata: Libellulidae), 41
individuals were collected from 14 sites in the Tono area of Gifu Prefecture, Japan (TGJ group). We
amplified a partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene (658 bp.)from the TGJ group and compared it with the same sequence of 68 N. pygmaea specimens were collectedfrom 6 sites, South Korea (SK group), to analyze the genetic diversity. The TGJ group comprised four
haplotypes (TGJ01–04), all of which differed from those in the SK group. Neighbor-joining phylogenetic
tree generated based on the COI sequences revealed divergence between the two groups. The distributionof the 10 haplotypes in the SK group was random and did not vary with distance or among the geographic
regions in South Korea, but this pattern of genetic distribution was not observed in our study. Rather,although the distribution of the TGJ01 haplotype was ubiquitously in the Tono area, the remaining
haplotypes (TGJ02, TGJ03 and TGJ04) exhibited localized distributions. Although our study was limitedby the number of individuals and the size of the area surveyed, we were able to clarify the phylogeny andgenetic variation in N. pygmaea in the Tono area of Gifu Prefecture, Japan.
Key words: Nannophya pygmaea, mitochondrial COI gene, genetic diversity,(Received: 4 April 2016; Accepted: 5 September 2016)negative influence of the populations of this species and canact to decrease overall genetic diversity.
For example, in South Korea, N. pygmaea is listed as asecond-degree endangered wild animal, generally due tothe same environmental problems that exist in Japan (Kimet al., 2007). Given this background, Kim et al. (2007)
sequenced a region of the mitochondrial cytochrome coxidase subunit I (COI) gene from 68 specimens collected
in 6 sites in South Korea. Their study showed that thepopulations in the southern part of South Korea exhibited
higher levels of genetic diversity compared to regions
elsewhere in the country. Moreover, phylogenetic analysis
and uncorrected pairwise distance estimates showed thatgenetic diversity estimates were relatively low in this species(Kim et al., 2007). For the long-term conservation of thespecies at a national scale, Kim et al. (2007) proposed that

Introduction
Nannophya pygmaea (Odonata: Libellulidae), one of
the smallest dragonflies in the world, is distributed from
China to Korea and Japan, through Southeast Asia, andAustralia (Ishida et al., 1988). In Japan, this species iswidely distributed throughout the Japanese mainland and islisted as an endangered species in some prefectures (Ishidaet al., 1988). The habitats of N. pygmaea include specificmarshes that are too shallow to support fish species thatprey on dragonfly larvae. Since N. pygmaea adults remain
at the same marsh after emergence (Ishida et al., 1988), thespecies is well suited for use as an environmental indicatorspecies. However, populations of N. pygmaea are threatenedby extensive habitat destruction, mainly due to factors suchas urbanization, and water pollution. These threats have a1)

College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan, 2) Collegeof Contemporary Education, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan (Corresponding Author:
Motoyasu MINAMI, E-mail: minami@isc.chubu.ac.jp)

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Motoyasu MINAMI et al.

the populations in the southern part of the country required
more efforts to preserve species-level genetic diversity.
While some studies have been conducted on the habitat
preference (Yoshida et al., 2004) and distribution (Ishidaet al., 1988) of this species in Japan, no studies haveexamined the genetic diversity of N. pygmaea to date.
In order to clarify the genetic diversity of N. pygmaea inJapan, we therefore investigated the partial sequences ofthe mitochondrial COI gene region of the populations of
this species in the Tono area of Gifu Prefecture of Japan.
Moreover, the sequence data were then used to estimate thegenetic diversity within a target population and to perform amolecular phylogenetic analysis using the specimens fromthe Japanese populations (this study) and South Korean
populations (Kim et al., 2007).

Table 1. Frequencies of 4 haplotypes of Nannophya pygmaea
in 14 sampling sites on the Tono area of Gifu Prefecture,
Japan.
Number of haplotype

Locality

TGJ01

TGJ02

TGJ03

1‐

‐

11‐

‐

‐

1‐

‐

‐

3‐

‐

‐

Kita-machi, Tajimi(35°20', 137°06')

4‐

‐

‐

Hoshigadai, Tajimi(35°19', 137°08')

3‐

‐

‐

Izumikitayama-cho, Toki
(35°23', 137°11')

3

1‐

‐

Tsurusato-cho, Toki
(35°16', 137°13')

3‐

‐

‐

Kamado-cho, Mizunami(35°25', 137°17')

4‐

‐

‐

Kitaogi-cho, Tajimi(four different sampling sites)(35°20', 137°02')

Takenami-cho, Ena(four different sampling sites)(35°25', 137°21')
Naegi, Nakatsugawa(35°32', 137°28')
Total

TGJ04

4‐

2‐

2‐‐‐

3‐

1‐

1‐

1‐

2‐

‐

‐

35141

Materials and Methods
We collected 41 N. pygmaea specimens from 14 sites in theTono area of Gifu Prefecture (Table 1). The left middle legsof N. pygmaea specimens were removed for DNA analysis,
where after the specimens were released at the collectionsites. The tissues thus collected were stored at −20°C until

Table 2. The haplotypes defined by polymorphic nucleotide sites in a partial sequence of the mitochondrial COI genes ofNannophya pygmaea on the Tono area of Gifu Prefecture, Japan, and South Korea.
HaplotypeNucleotide potition 1)
1928

109

214

271

352

367

478

484

586

589

616

619

625

634

TGJ01

CAAGAATCAGTTTGC

TGJ02***A***********TGJ03****C***T******

TGJ04

*****

GCT

*******BARNP01

T*************T

BARNP02*G************T

BARNP03**G***********T

BARNP04***

A**********T

BARNP05*********

A

****.

BARNP06**********C***TBARNP07***********C**TBARNP08

*****

***

****

C*

TBARNP09*************ATBARNP10**************TTono area2)South Korea

3)

Asterisk indicates the identical nucleotide to which of TGJ01.
1) Variation site are shown as nucleotide positions from 5' end of PCR amplified region, except forward primer sequence.
2) TGJ01 - 04 were confirmed on Tono area of Gifu Prefecture in Japan
3) BARNP01 - 10 were confirmed in South Korea (Kim et al., 2007)Ⓒ2016

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Molecular phylogenetic analysis of Nannophya pygmaea

total genomic DNA was extracted using a DNeasy Bloodand Tissue Kit (Qiagen, Germany) and purified using aGeneClean Spin Kit (MP Biomedicals, CA).
PCR was performed in 50 μl reaction mixtures with0.5 units of KOD-Plus-DNA Polymerase (Toyobo,Japan) and 0.2 µM of each primer, according to themanufacturer’s instructions. The primer sequences forthe regions flanking the COI locus were (forward) 5′-GG
TCAACAAATCATAAAGATATTGG-3′, and (reverse)
5′-TAAACTTCAGGGTGACCA AAAAATCA-3′ (Murajiet al., 2001). PCR reactions were performed using a
DNA Thermal Cycler (GeneAmp PCR System 9700,Applied Biosystems, Foster City, CA) using an initialdenaturation step of 94°C for 2 min, followed by 40 cyclesof denaturation at 94°C for 15 s, annealing at 47°C for 30s, extension at 68°C for 90 s, and a final extension stepof 68°C for 90 s. All PCR products were purified using aQIAquick PCR Purification Kit (Qiagen) and subjected todye-terminator cycle sequencing using a DTCS Quick Start
Mix (Beckman Coulter, CA) with an automatic sequencer(CEQ 2000XL, Beckman Coulter, Inc.).
The COI sequences were aligned with the publishedsequences from South Korean species (Kim et al., 2007)
using MEGA5.05 software (Tamura et al., 2011), which was
also used to conduct phylogenetic analyses. Specifically,neighbor-joining (NJ) tree were generated from the COI
sequences using the Maximum Composite Likelihood
algorithm in MEGA5.05. Bootstrap analysis was performed
to estimate the robustness of each node in the phylogeny(10,000 replicates). The partial sequence of Orthetrum
triangulare melania COI (Accession No. AB126005) wasused as an outgroup.

Results and Discussion
The alignment of a continuous stretch of 658 bp of the COI
sequences revealed nucleotide variability at six positions. A
total of four haplotypes (TGJ01–04) was obtained for the N.
pygmaea specimens from 14 sites in the Tono area of Gifu
Prefecture (Table 2). The TGJ01 haplotype was identified in
85% (35/41) of all of the specimens in the Tono area of Gifu
Prefecture (TGJ) group, and is distributed throughout theTono area of Gifu Prefecture. The distribution of the TGJ01
haplotype appeared to be ubiquitously and independentof both distance and geographic location in the Tono
area. However, the remaining three haplotypes (TGJ02,TGJ03 and TGJ04) showed evidence of local adaptation.
In particular, the TGJ02 and TGJ04 haplotypes were both
only observed in one individual each in Izumikitayamacho in Toki and Kitaogi-cho in Tajimi, respectively
BARNP03
BARNP0937

BARNP06

25

BARNP0730

BARNP10BARNP08

4743

SK group

BARNP02BARNP01
BARNP04
BARNP05
TGJ0262

TGJ04

4194

TGJ01

TGJ group

TGJ03
Orthetrum triangulare melania（Accession No. AB126005）

0.02
Neighbor-joiningtree
trees based
based onon
mitochondrial
COI genepartialsequences
Nannophya pygmaea
on Tono area of Gifu Prefecture,
Fig.1. Fig.1.
Neighbor-joining
mitochondrial
COIgenepartialof sequencesof Nannophya
Japan (TGJ group) and South Korea (SK group).
pygmaeaonTonoareaofGifu
Prefecture,
Japan(TGJgroup)
andSouthKorea(SKgroup).
Orthetrum triangulare melania （Accession No. AB126005） was used as an outgroup. Numbers beside the branches indicate bootstrapvalues
Orthetrum
triangularemelania（AccessionNo. AB126005）
wasalphabet
used asoutgroup.
Numbersin 10,000replicates. Branch
lengths
represent nucleotidesubstitutions. The
andan
numberwere shown
each haplotype name (shownTable
1 and 2). Sequence
databootstrapvaluesof SK group was quoted
from the reportof Kim etBranchal. (2007).lengths represent
besideinthebranches
indicatein 10,000replicates.

nucleotide substitutions. The alphabet and number were shown each haplotype name (shown in
Table 1 and 2). Sequence data of SK group was quoted from the report of Kim et al. (2007).

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Motoyasu MINAMI et al.

(Table 1). The TGJ03 haplotype was confirmed in fourindividuals in Takenami-cho in Ena (Table 1). Although thedifferent geographic populations of N. pygmaea in South
Korea contained 10 haplotypes, the distribution of these
haplotypes did not show any variation with distance oramong geographic regions (Kim et al., 2007). This genetic
pattern was not comparable to that observed in our study,as the regional genetic variation observed in N. pygmaea
specimens from the Tono area was less than that observed
among specimens from South Korea.
Sequence alignment of the four N. pygmaea haplotypes(TGJ01–04) identified in this study and the 10 N. pygmaea
haplotypes (BARNP01–10) identified in South Korea (Kimet al., 2007) revealed 15 variable nucleotide positions.
In addition, NJ analysis revealed that the N. pygmaea
sequences formed two distinct clades (TGJ and SK groups)(Fig. 1). We identified the intraspecific COI variants inboth groups and found that the four endemic haplotypes
comprising the TGJ group were responsible for theseparation of the clades.
Although somewhat limited in geographical scale, weconducted this preliminary case study to clarify the genetic
diversity and regional genetic variation of N. pygmaea
populations in the Tono area of Gifu Prefecture. Futurestudies will therefore involve genetic surveys of N. pygmaea
populations in other regions so that we can compare suchfindings with those of this study.

phylogenetic analyses in anthocorid bugs (Hemiptera:Anthocoridae), Japan Agricultural Research Quarterly,
35: 85-90.
Tamura, K., D. Peterson, G. Stecher, M. Nei and S. Kumar(2011): MEGA5: Molecular evolutionary genetics
analysis using maximum likelihood, evolutionary
distance, and maximum parsimony methods. MolecularBiology and Evolution, 28: 2731-2739.
Yoshida, S., Y. Minami and T. Ueda, T (2004): Water
chemistry of several habitats of a tiny dragonfly,
Nannophya pygmaea Rambur. Japanese Journal ofEnvironmental Entomology and Zoology, 15: 13-17. (inJapanese with English summary)(Editor: Dr. Kentaro NOZAKI, School of Education,
Sugiyama Jogakuen University)摘要

岐阜県東濃地方と韓国に生息するハッチョウトンボ（Nannophya pygmaea）の遺伝的多様性についての比較
南基泰1)，味岡ゆい2)，中村早耶香1)
ハッチョウトンボのミトコンドリア COI 遺伝子部分領域（658塩基）について，岐阜県東濃地方14地点 41個体（東濃グループ）の DNA 配列を決定した．決定された東濃グループの COI と，韓国で報告された 6地点 68個体（韓国グループ）

Acknowledgmentsの COI について分子系統学的解析を行った．東濃グループ

The authors thank Mr. Hiroyuki ARAKAI in the Tajimi
Naturalist Club for valuable information about habitats ofN. pygmaea, and Mr. Michio SAWAI, Ms. Akari TOMURA Ms.
Mio SHINODA and Mr. Hironori MIZUNO for technical support.

ハプロタイプ（TGJ01-04）が確認された．そして，近隣接合からは，韓国グループからは確認されていない新たな 4つの法による分子系統樹を構築した結果，東濃グループと韓国グループは異なる 2つのクレードに分かれた．韓国グループの10のハプロタイプは，韓国国内にランダムに分布し，地理的局在性は認められないと報告されている．一方，東濃グルー
プでは最も多く確認された TGJ01（85%）については東濃全References
Ishida, S., K. Ishida, K. Kojima and M. Sugiura (1988):
Illustrated guide for identification of the JapaneseOdonata. Tokai University Press, Tokyo. (in Japanese)
Kim, K.G., S.K. Jang, D.W. Park, M.Y. Hong, K.H. Oh,K.Y. Kim, J.S. Hwang, Y.S. Han and I. Kim (2007):
Mitochondrial DNA sequence variation of the tiny
dragonfly, Nannophya pygmaea (Odonata: Libellulidae),International Journal of Industrial Entomology, 15: 47-58.
Muraji, M., K. Kawasaki and T. Shimizu (2001): Nucleotide
sequence variation and use of mitochondrial DNA forⒸ2016

The Tokai Branch of the Japanese Society of Limnology

体にランダムに分布していたが，残りの 3つのハプロタイプ（TGJ02, TGJ03 and TGJ04）については地域局在性が認められた．本報告では，調査域が東濃全域に及んでいないが，東濃のハッチョウトンボは韓国のものと分子系統的に異なり，遺伝的変異に地理的局在性があることが明らかとなった．
キーワード：ハッチョウトンボ，
ミトコンドリア COI 遺伝子，
遺伝的多様性1)

中部大学応用生物学部環境生物科学科，487-8501愛知県春日井市松本町 1200番地
2)
中部大学現代教育学部児童教育学科，487-8501愛知県春日井市松本町 1200番地（連絡著者：南基泰 E-mail:minami@isc.chubu.ac.jp）

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