kazumi tanida , nobuyoshi shimizu, mikio kato

DNA Barcodes 2016; 4: 1–19

families within the orders but were useful in identifying species (i.e., DNA barcoding). In contrast, the more conserved 18S rRNA gene was adequately informative for elucidating inter-family divergence, but not suitable for species identification.

Keywords: COI; gene phylogeny; histone; mayfly; rRNA; species identification

1 IntroductionAquatic insects comprise a taxonomically diverse community in rivers. Some taxonomic groups (taxa) are used as indicators for monitoring ecosystem health [1]. Despite their ecological importance, some groups (e.g., mayflies, Ephemeroptera) are often neglected in identification at the species-level in routine ecological assessments, and even in detailed research projects. Mayflies serve as indicators for ecological integrity because they are sensitive to environmental conditions such as water quality and flow characteristics [2-4]. Because of their small size, high abundance, diversity, and complex taxonomy, even experienced taxonomists find difficulty in species identification, especially when handling immature stages or damaged specimens. As an alternative to the classical identification based on morphological characters, DNA-based identification had been proposed [5] and DNA barcoding (originally proposed by Hebert et al. [6]) has become standard for characterizing a variety of taxonomic groups including mayflies. DNA barcoding uses a matching analysis of a short genetic marker (DNA barcode) to DNA databases of identified specimens. The utility and reliability of DNA barcoding depend upon the quality of DNA databases in which the annotated sequence data are registered. While 142 species of Japanese mayflies have been described from 39 genera in 13 families [7], only 17 species in 8 families of

DOI 10.1515/dna-2016-0001Received August 18, 2015; accepted February 29, 2016

Abstract: We have determined nucleotide sequences of three genes encoding histone H3, 18S rRNA, and mitochondrial cytochrome oxidase subunit I (COI) from the mayfly community in Eastern Asia. All sequence data collected from 303 specimens comprising 80 taxa (63 identified to species, 14 to genus, and 3 to family) were deposited in GenBank/ EMBL/ DDBJ International Databases for further expansion of DNA barcoding projects of mayflies. Phylogenetic analysis based on the neighbor-joining method among the three genes found that COI and H3 genes had relatively high evolutionary rates, and were not suitable in inferring phylogenetic relationships among

Research Article Open Access

© 2016 Kei Wakimura, et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.

Kei Wakimura, Yasuhiro Takemon, Atsushi Takayanagi, Shin-ichi Ishiwata, Kozo Watanabe, Kazumi Tanida#, Nobuyoshi Shimizu, Mikio Kato*

Characterization of genes for histone H3, 18S rRNA, and cytochrome oxidase subunit I of East Asian mayflies (Ephemeroptera)

Article note: N. Shimizu, who designed the work and was responsible for DNA sequencing, passed away on June 5, 2015, after his approval for submitting the manuscript on May 26, 2015. This paper is dedicated to him.

*Corresponding author Mikio Kato, Biology Laboratory, Faculty of Liberal Arts and Sciences, Osaka Prefecture University, 1-1 Gakuencho, Naka-ku, Sakai 599-8531, Japan, E-mail: [email protected] Kei Wakimura, Biology Laboratory, Faculty of Liberal Arts and Sciences, Osaka Prefecture University, 1-1 Gakuencho, Naka-ku, Sakai 599-8531, JapanYasuhiro Takemon, Disaster Prevention Research Institute, Kyoto University, Uji 611-0011, JapanAtsushi Takayanagi, Nobuyoshi Shimizu, Advance Research Center for Genome Super Power, Keio University, 2 Ohkubo, Tsukuba 300-2611, JapanShin-ichi Ishiwata, Department of Applied Chemistry, Kanagawa Institute of Technology, 1030 Shimo-Ogino, Atsugi 243-0292, JapanKozo Watanabe, Department of Civil and Environmental Engineering, Ehime University, Bunkyo-cho 3, Matsuyama 790-8577, JapanKazumi Tanida, Mikio Kato, Department of Biological Sciences, Graduate School of Science, Osaka Prefecture University, 1-1 Gaku-encho, Naka-ku, Sakai 599-8531, Japan#Present address: Osaka Museum of Natural History, Nagai Park, Higashi-sumiyoshi, Osaka, 546-0034 Japan

2 K. Wakimura, et al.

Japanese mayflies’ DNA sequences appeared in GenBank/ EMBL/ DDBJ International DNA databases (http://www.ncbi.nlm.nih.gov/genbank/) when searched by the terms “Ephemeroptera” + “Japan” + NOT “Osaka Prefecture” (accessed 25 May, 2015). To accelerate DNA barcoding in surveying the mayfly biodiversity in the East Asian area, more annotated sequences must be registered to the databases.

The present work aims to collect molecular data of East Asian mayflies from three genes that are widely used for taxonomic and evolutionary studies; a 658-bp partial coding region of cytochrome oxidase subunit I (COI) as suggested by Consortium for the Barcode of Life (CBOL, http://www.barcodeoflife.org/), a 328-bp partial coding region of histone 3 (H3), and a partial sequence of 18S rRNA (about 550-bp). All genes have been utilized to deduce the phylogenetic relationship among Ephemeroptera species [8]. For 18S rRNA, we chose the target region covering V4 and V5 stem-loop structures because of their high level of genetic variation within the order Ephemeroptera [9].

2 Materials and methods

2.1 Sample specimens

We collected 303 specimens (109 imagoes, 148 nymphs, 46 subimagoes) from 80 taxa which were composed of 63 species, 14 genera (unidentified specific name), and 3 families (unidentified genus name) for sequencing analysis (Table 1). In total, there were 33 genera belonging to 11 families. These samples were collected from Honshu, Kyushu and Ryukyu Islands of Japan and Taiwan in the years of 1997-2014. Specimens were identified by the authors before DNA extraction. To avoid false-positive identification in DNA barcoding, only accurately identified specimens must be registered with full scientific names in the DNA databases. Therefore, taking a conservative approach, we did not assign scientific names to the specimens that could not be identified unambiguously due to damage or their early developmental stages.

Fresh specimens were dissected to isolate DNA samples, while some were stored at -80°C, or in absolute ethanol at room temperature until the time of dissection. DNA was extracted from wing muscles at mid-thorax (imago/subimago females and males), sperm vesicles (imago/subimago males), hemolymph containing several types of somatic cells (nymphs), whole thorax (small individuals), or legs (relatively large individuals) of the

specimens, followed by a conventional proteinase-phenol extraction protocol [10]. Genitalia and heads parts were kept intact during dissection as voucher specimens, and stored in absolute ethanol. However, the genitalium of some small specimens was damaged during the isolation of sperm vesicles. For a few small specimens (e.g., Baetidae), a whole body was mashed in a micro-centrifuge tube prior to DNA extraction.

All the information about the samples such as developmental stage, sex, date of collection, locality, by whom collected and identified, source organ for DNA isolation, and raw data of DNA sequencing were recorded. These are available from the corresponding author (Kato) upon request. DNA samples were stored in the refrigerator and insect specimens were kept at room temperature in absolute ethanol. All the samples used in this study are safely kept in the corresponding author’s laboratory (Osaka Prefecture University) except those safely stored in Takemon’s laboratory (Kyoto University) (specimen vouchers KU_Rj2008-60 ~ 69).

2.2 DNA analyses

PCR was performed using a set of DNA primers designed for amplifying the target genes (Table 2), with either Sensizyme® HotStart Taq Premix (RBC Bioscience) or AmpliTaq Gold® 360 Master Mix (Life Technologies), depending on the availability. Amplified DNA fragments were purified using HiYield™ Gel/PCR DNA Fragments Extraction Kit (RBC Bioscience) and sequenced from both forward and reverse directions using BigDye® Terminator v3.1 (Life Technologies) and an ABI3730xl DNA Analyzer (Applied Biosystems - Life Technologies). Forward and reverse sequencing reads were assembled, and aligned using BioEdit Sequence Alignment Editor version 7.1.3.0 [11]. Phylogenetic trees for each of the three genes were reconstructed based on the neighbor-joining method [12] using “Dnadist” with the option F84 model and “Neighbor” implemented in PHYLIP [13] with outgroup sequences (accession numbers: KC135891 for COI, AY870292 for histone H3, EF680326 for 18S rRNA). Tree topologies were examined by bootstrapping sequences of 1000 pseudoreplicates using “Seqboot” in PHYLIP. For the tree reconstructions, we used only sequence data that has a complete set of the three genes for simple comparison of tree topologies among different genes. Incomplete data sets (i.e., specimens of which only one or two genes out of three were determined) were not incorporated to the analysis.

DNA barcodes of Japanese mayflies 3

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

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e da

ta a

nd s

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e in

form

atio

n.

Fam

ily n

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Spec

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nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

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COI

Spe

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en vo

uche

r

Hept

agen

iidae

Afro

nuru

s hy

alin

usim

ago

sper

m ve

sicl

esTa

iwan

27 S

ep, 2

011

JQ65

0220

KF56

2953

KF56

3027

OPU_

BS_E

h201

1-51

6

Afro

nuru

s hy

alin

usim

ago

win

g m

uscl

eTa

iwan

27 S

ep, 2

011

KF56

3028

OPU_

BS_E

h201

1-51

7

Afro

nuru

s hy

alin

usim

ago

win

g m

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eTa

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27 S

ep, 2

011

JQ65

0221

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BS_E

h201

1-51

8

Afro

nuru

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mph

hem

olym

phW

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Japa

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May

, 201

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5628

67

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011-

168

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Nov

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011-

625

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25 N

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011-

627

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1-62

8

Blep

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Kyot

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17 Fe

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0

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Feb,

201

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96

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JQ65

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3023

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2011

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14 A

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3024

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2011

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May

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30

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Apr

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197

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012-

198

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Apr

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2KF

5629

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012-

199

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mph

who

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Wak

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a, Ja

pan

15 M

ay, 2

011

KP97

0811

JQ65

0162

OP

U_BS

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011-

164


Fam

ily n

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nam

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tion

Colle

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te18

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Spe

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Apr,

2012

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2012

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2012

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2013

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2013

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2013

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2011

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an27

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57

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an27

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0687

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3-58

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rus

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ago

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g m

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010

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Apr,

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6501

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ago

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m ve

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Apr

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6803

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ago

win

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6502

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010-

4

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Apr

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m ve

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0174

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BS_E

l201

1-20

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rus

latif

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ago

sper

m ve

sicl

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ra, J

apan

14 A

pr, 2

011

JQ65

0175

JQ65

0119

KF56

3019

OPU_

BS_E

l201

1-21

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rus

latif

oliu

mim

ago

sper

m ve

sicl

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ra, J

apan

14 A

pr, 2

011

JQ65

0176

JQ65

0130

KF56

3020

OPU_

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l201

1-22

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latif

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mim

ago

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m ve

sicl

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ra, J

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14 A

pr, 2

011

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2869

OPU_

BS_E

l201

1-23

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rus

latif

oliu

mim

ago

sper

m ve

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ra, J

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14 A

pr, 2

011

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0177

JQ65

0131

JQ65

5115

OPU_

BS_E

l201

1-24

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Epeo

rus

latif

oliu

mim

ago

win

g m

uscl

eNa

ra, J

apan

14 A

pr, 2

011

KF56

2870

OPU_

BS_E

l201

1-25

Epeo

rus

latif

oliu

mim

ago

win

g m

uscl

eNa

ra, J

apan

14 A

pr, 2

011

KF56

2871

JQ65

0154

OPU_

BS_E

l201

1-26

Epeo

rus

latif

oliu

mim

ago

sper

m ve

sicl

esNa

ra, J

apan

14 A

pr, 2

011

JQ65

0178

JQ65

0132

OPU_

BS_E

l201

1-27

Epeo

rus

latif

oliu

mim

ago

sper

m ve

sicl

esNa

ra, J

apan

14 A

pr, 2

011

KF56

2872

OPU_

BS_E

l201

1-28

Epeo

rus

latif

oliu

mim

ago

sper

m ve

sicl

esNa

ra, J

apan

14 A

pr, 2

011

JQ65

0155

OPU_

BS_E

l201

1-29

Epeo

rus

latif

oliu

mim

ago

sper

m ve

sicl

esNa

ra, J

apan

14 A

pr, 2

011

JQ65

0156

OPU_

BS_E

l201

1-30

Epeo

rus

latif

oliu

mim

ago

sper

m ve

sicl

esNa

ra, J

apan

14 A

pr, 2

011

JQ65

0157

KF56

3022

OPU_

BS_E

l201

1-56

Epeo

rus

latif

oliu

msu

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0758

OPU_

BS_E

l201

3-1

Epeo

rus

latif

oliu

msu

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0759

OPU_

BS_E

l201

3-2

Epeo

rus

latif

oliu

msu

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0760

OPU_

BS_E

l201

3-4

Epeo

rus

latif

oliu

msu

bim

ago

sper

m ve

sicl

esNa

ra, J

apan

27 A

pr, 2

013

KP97

0761

OPU_

BS_E

l201

3-5

Epeo

rus

latif

oliu

msu

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0700

OPU_

BS_E

l201

3-14

3

Epeo

rus

latif

oliu

mim

ago

leg

Wak

ayam

a, Ja

pan

11 M

ay, 2

014

KP97

0713

OPU_

BS_E

l201

4-1

Epeo

rus

latif

oliu

mim

ago

leg

Wak

ayam

a, Ja

pan

11 M

ay, 2

014

KP97

0714

OPU_

BS_E

l201

4-2

Epeo

rus

napa

eus

imag

osp

erm

vesi

cles

Nara

, Jap

an9

May

, 200

9GU

3541

59

GU35

4163

Ena2

009-

125-

Sann

okou

Epeo

rus

napa

eus

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KF56

2896

OPU_

BS_E

n201

2-25

1

Epeo

rus

napa

eus

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KF56

2897

OPU_

BS_E

n201

2-25

2

Epeo

rus

napa

eus

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KF56

2898

OPU_

BS_E

n201

2-25

3

Epeo

rus

sp.

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KP97

0812

KP97

0755

OPU_

BS_E

2012

-235

Epeo

rus

sp.

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KP97

0756

OPU_

BS_E

2012

-236

Hept

agen

ia k

ihad

any

mph

hem

olym

phSh

iga,

Japa

nM

ar, 2

012

KF56

2875

OPU_

BS_H

k201

2-24

Proe

peor

us n

ippo

nicu

sim

ago

sper

m ve

sicl

esNa

ra, J

apan

9 M

ay, 2

009

GU35

4160

GU35

4164

Eni2

009-

131-

Naka

igaw

aRh

ithro

gena

japo

nica

imag

ow

ing

mus

cle

Kyot

o, Ja

pan

27 A

pr, 2

008

JQ31

9378

KU_R

j200

8-63

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esKy

oto,

Japa

n27

Apr

, 200

8JQ

3193

79KU

_Rj2

008-

64

Rhith

roge

na ja

poni

caim

ago

win

g m

uscl

eKy

oto,

Japa

n27

Apr

, 200

8JQ

3193

80KU

_Rj2

008-

65

Rhith

roge

na ja

poni

caim

ago

win

g m

uscl

eKy

oto,

Japa

n27

Apr

, 200

8JQ

3193

81KU

_Rj2

008-

67

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esKy

oto,

Japa

n27

Apr

, 200

8JQ

3193

82KU

_Rj2

008-

68

Rhith

roge

na ja

poni

caim

ago

win

g m

uscl

eKy

oto,

Japa

n27

Apr

, 200

8JQ

3193

83KU

_Rj2

008-

69

Rhith

roge

na ja

poni

cany

mph

who

le b

ody

Nara

, Jap

an14

Dec

, 201

2KF

5628

94KF

5629

76OP

U_BS

_Rj2

012-

241

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Rhith

roge

na ja

poni

cany

mph

who

le b

ody

Nara

, Jap

an14

Dec

, 201

2KF

5628

95KF

5629

77OP

U_BS

_Rj2

012-

242

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n24

Apr

, 200

5JQ

3193

84OP

U_Rj

2005

-02

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n24

Apr

, 200

5JQ

3193

85OP

U_Rj

2005

-07

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n24

Apr

, 200

5JQ

3193

86OP

U_Rj

2005

-10

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n25

Apr

, 200

4EF

6803

32AY

8503

72RJ

2004

Niug

awa1

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esNa

ra, J

apan

25 A

pr, 2

009

GU35

4147

Rja2

009-

01-O

osak

o-U

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esNa

ra, J

apan

25 A

pr, 2

009

GU35

4148

Rja2

009-

02-O

osak

o-U

Rhith

roge

na ja

poni

caim

ago

sper

m ve

sicl

esNa

ra, J

apan

25 A

pr, 2

009

GU35

4149

Rja2

009-

03-O

osak

o-U

Rhith

roge

na m

inaz

uki

nym

phhe

mol

ymph

Okin

awa,

Japa

n22

Apr

, 201

2KF

5629

34KF

5630

58OP

U_BS

_Rm

2012

-216

Rhith

roge

na m

inaz

uki

nym

phw

hole

bod

yOk

inaw

a, Ja

pan

22 A

pr, 2

012

KF56

2935

OPU_

BS_R

m20

12-2

17

Rhith

roge

na m

inaz

uki

nym

phw

hole

bod

yOk

inaw

a, Ja

pan

23 A

pr, 2

012

KF56

2936

OPU_

BS_R

m20

12-2

18

Rhith

roge

na ta

teya

man

aim

ago

sper

m ve

sicl

esKa

naga

wa,

Japa

n16

Nov

, 201

1

JQ65

0139

OP

U_BS

_Rt2

011-

601

Rhith

roge

na ta

teya

man

aim

ago

win

g m

uscl

eKa

naga

wa,

Japa

n16

Nov

, 201

1JQ

6501

40OP

U_BS

_Rt2

011-

602

Rhith

roge

na ta

teya

man

asu

bim

ago

win

g m

uscl

eKa

naga

wa,

Japa

n16

Nov

, 201

1JQ

6501

98JQ

6501

41

OPU_

BS_R

t201

1-60

3

Rhith

roge

na

tetra

punc

tiger

any

mph

leg

Saita

ma,

Japa

n6

Dec,

200

8KP

9707

86KP

9707

06OP

U_BS

_Rt2

013-

358

Rhith

roge

na

tetra

punc

tiger

any

mph

leg

Saita

ma,

Japa

n6

Dec,

200

8KP

9707

87KP

9707

07OP

U_BS

_Rt2

013-

359

Rhith

roge

na s

p.

imag

osp

erm

vesi

cles

Nara

, Jap

an25

Apr

, 200

9GU

3541

53GQ

4339

97

Rxx2

009-

08-O

osak

o-U

Rhith

roge

na s

p.su

bim

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n24

Apr

, 201

0JQ

6502

02OP

U_BS

_R20

10-1

1

Rhith

roge

na s

p.su

bim

ago

win

g m

uscl

eW

akay

ama,

Japa

n24

Apr

, 201

0KF

5630

12OP

U_BS

_R20

10-1

5

Rhith

roge

na s

p.su

bim

ago

win

g m

uscl

eW

akay

ama,

Japa

n24

Apr

, 201

0JQ

6501

46KF

5630

13OP

U_BS

_R20

10-1

6

Rhith

roge

na s

p.su

bim

ago

win

g m

uscl

eW

akay

ama,

Japa

n24

Apr

, 201

0JQ

6502

03KF

5630

10OP

U_BS

_R20

10-1

2

Rhith

roge

na s

p.su

bim

ago

win

g m

uscl

eW

akay

ama,

Japa

n24

Apr

, 201

0KF

5630

14OP

U_BS

_R20

10-1

9

Rhith

roge

na s

p.im

ago

win

g m

uscl

eW

akay

ama,

Japa

n5

May

, 201

2KF

5629

62OP

U_BS

_R20

12-1

02

Rhith

roge

na s

p.im

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n5

May

, 201

2KF

5629

11KF

5629

64OP

U_BS

_R20

12-1

53

Rhith

roge

na s

p.im

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n5

May

, 201

2KF

5629

12KF

5629

65OP

U_BS

_R20

12-1

54

Rhith

roge

na s

p.im

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n5

May

, 201

2KF

5629

13KF

5629

66OP

U_BS

_R20

12-1

55

Rhith

roge

na s

p.im

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n5

May

, 201

2KF

5629

67OP

U_BS

_R20

12-1

56

Rhith

roge

na s

p.im

ago

win

g m

uscl

eW

akay

ama,

Japa

n5

May

, 201

2KF

5628

77KF

5629

68OP

U_BS

_R20

12-1

57

Rhith

roge

na s

p.im

ago

sper

m ve

sicl

esW

akay

ama,

Japa

n5

May

, 201

2KF

5629

14KF

5629

69

OPU_

BS_R

2012

-158

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Hept

agen

iidae

sp.

im

ago

win

g m

uscl

eW

akay

ama,

Japa

n28

Apr

, 201

3KP

9707

81OP

U_BS

_H20

13-2

35

Ephe

mer

ellid

ae

Cinc

ticos

tella

elo

ngat

ula

subi

mag

ow

ing

mus

cle

Nara

, Jap

an15

Apr

, 201

1JQ

6501

85JQ

6501

36OP

U_BS

_Ce2

011-

98

Cinc

ticos

tella

elo

ngat

ula

subi

mag

osp

erm

vesi

cles

Nara

, Jap

an15

Apr

, 201

1JQ

6501

86JQ

6501

21OP

U_BS

_Ce2

011-

99

Cinc

ticos

tella

elo

ngat

ula

subi

mag

osp

erm

vesi

cles

Nara

, Jap

an15

Apr

, 201

1JQ

6501

91JQ

6501

23OP

U_BS

_Ce2

011-

128

Cinc

ticos

tella

nig

ra

imag

ow

ing

mus

cle

Nara

, Jap

an8

May

, 201

0JQ

6502

10

OP

U_BS

_Cn2

010-

363

Cinc

ticos

tella

nig

ra

nym

phhe

mol

ymph

Wak

ayam

a, Ja

pan

15 M

ay, 2

011

JQ65

0218

OPU_

BS_

Cn20

11-1

71

Cinc

ticos

tella

nig

ra

nym

phhe

mol

ymph

Nara

, Jap

an14

May

, 201

1JQ

6501

94JQ

6501

26

OPU_

BS_C

n201

1-30

8

Cinc

ticos

tella

orie

ntal

isny

mph

hem

olym

phNa

ra, J

apan

14 M

ay, 2

011

JQ65

0195

JQ65

0127

OPU_

BS_C

o201

1-30

9

Cinc

ticos

tella

sp.

imag

ow

ing

mus

cle

Nara

, Jap

an15

Apr

, 201

1JQ

6501

89JQ

6501

61

OPU_

BS_C

2011

-113

Cinc

ticos

tella

sp.

imag

ow

ing

mus

cle

Nara

, Jap

an15

Apr

, 201

1

KF56

3005

OP

U_BS

_C20

11-1

17

Drun

ella

bas

alis

ny

mph

hem

olym

phKa

naga

wa,

Japa

nM

ar, 2

012

KF56

2885

KF56

2974

KF56

3033

OPU_

BS_D

b201

2-1

Drun

ella

bas

alis

ny

mph

hem

olym

phKa

naga

wa,

Japa

nM

ar, 2

012

KF56

2886

KF56

3034

OPU_

BS_D

b201

2-2

Drun

ella

bas

alis

ny

mph

hem

olym

phKa

naga

wa,

Japa

nM

ar, 2

012

KF56

3035

OPU_

BS_D

b201

2-5

Drun

ella

bas

alis

ny

mph

hem

olym

phKa

naga

wa,

Japa

nM

ar, 2

012

KF56

3036

OPU_

BS_D

b201

2-6

Drun

ella

bas

alis

ny

mph

hem

olym

phKa

naga

wa,

Japa

nM

ar, 2

012

KF56

3037

OPU_

BS_D

b201

2-7

Drun

ella

bas

alis

ny

mph

hem

olym

phFu

kuok

a, Ja

pan

16 A

pr, 2

012

KF56

3042

OPU_

BS_D

b201

2-10

0

Drun

ella

bas

alis

ny

mph

hem

olym

phFu

kuok

a, Ja

pan

16 A

pr, 2

012

KF56

3043

OPU_

BS_D

b201

2-10

1

Drun

ella

bas

alis

ny

mph

hem

olym

phFu

kuok

a, Ja

pan

16 A

pr, 2

012

KF56

3044

OPU_

BS_D

b201

2-16

0

Drun

ella

bas

alis

ny

mph

hem

olym

phFu

kuok

a, Ja

pan

16 A

pr, 2

012

KF56

3045

OPU_

BS_D

b201

2-16

1

Drun

ella

bas

alis

ny

mph

win

g m

uscl

eFu

kuok

a, Ja

pan

16 A

pr, 2

012

KF56

3046

OPU_

BS_D

b201

2-16

4

Drun

ella

cryp

tom

eria

nym

phw

ing

mus

cle

Wak

ayam

a, Ja

pan

5 M

ay, 2

012

KP97

0807

KP97

0751

OP

U_BS

_D20

12-2

22

Drun

ella

cryp

tom

eria

nym

phw

ing

mus

cle

Wak

ayam

a, Ja

pan

5 M

ay, 2

012

KP97

0808

KP97

0752

OPU_

BS_D

2012

-223

Drun

ella

cryp

tom

eria

nym

phw

ing

mus

cle

Wak

ayam

a, Ja

pan

5 M

ay, 2

012

KP97

0809

KP97

0753

OP

U_BS

_D20

12-2

24

Drun

ella

ishi

yam

ana

nym

phw

hole

bod

yW

akay

ama,

Japa

n15

May

, 201

1KF

5628

66JQ

6501

24OP

U_BS

_Di2

011-

165

Drun

ella

koh

noi

nym

phw

ing

mus

cle

Wak

ayam

a, Ja

pan

5 M

ay, 2

012

KF56

2937

KF56

3000

OP

U_BS

_Dk2

012-

220

Drun

ella

sac

halin

ensi

s ny

mph

hem

olym

phW

akay

ama,

Japa

n15

May

, 201

1KF

5628

68OP

U_BS

_Di2

011-

170

Drun

ella

sac

halin

ensi

s ny

mph

hem

olym

phKa

naga

wa,

Japa

nM

ar, 2

012

KF56

2902

KF56

2989

KF56

3040

OPU_

BS_D

s201

2-54

Drun

ella

sac

halin

ensi

s ny

mph

who

le b

ody

Kana

gaw

a, Ja

pan

Mar

, 201

2KF

5629

03KF

5629

90KF

5630

41OP

U_BS

_Ds2

012-

55

Drun

ella

tris

pina

ny

mph

hem

olym

phNa

ra, J

apan

14 M

ay, 2

011

JQ65

0193

OPU_

BS_D

t201

1-25

5

Drun

ella

sp.

nym

phw

hole

bod

yW

akay

ama,

Japa

n15

May

, 201

1KP

9708

06KP

9707

50OP

U_BS

_D20

11-1

66

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Drun

ella

sp.

nym

phw

ing

mus

cle

Kana

gaw

a, Ja

pan

Mar

, 201

2KF

5629

06KF

5629

60KF

5630

55OP

U_BS

_D20

12-9

6

Drun

ella

sp.

nym

phhe

mol

ymph

Kana

gaw

a, Ja

pan

Mar

, 201

2KF

5629

07KF

5629

61OP

U_BS

_D20

12-9

7

Drun

ella

sp.

nym

phhe

mol

ymph

Fuku

oka,

Japa

n15

Apr

, 201

2KF

5629

17OP

U_BS

_D20

12-1

78

Drun

ella

sp.

nym

phhe

mol

ymph

Fuku

oka,

Japa

n15

Apr

, 201

2KF

5629

18KF

5630

48OP

U_BS

_D20

12-1

79

Drun

ella

sp.

nym

phhe

mol

ymph

Fuku

oka,

Japa

n15

Apr

, 201

2KF

5629

19KF

5630

49OP

U_BS

_D20

12-1

80

Drun

ella

sp.

nym

phhe

mol

ymph

Fuku

oka,

Japa

n15

Apr

, 201

2KF

5629

20KF

5630

50OP

U_BS

_D20

12-1

81

Drun

ella

sp.

nym

phhe

mol

ymph

Fuku

oka,

Japa

n15

Apr

, 201

2KF

5629

21OP

U_BS

_D20

12-1

82

Epha

cere

lla lo

ngic

auda

ta

nym

phhe

mol

ymph

Shig

a, Ja

pan

Mar

, 201

2KF

5628

87KF

5629

82

OPU_

BS_E

l201

2-18

Ephe

mer

ella

ata

gosa

na

nym

phhe

mol

ymph

Shig

a, Ja

pan

Mar

, 201

2KF

5628

88KF

5629

83KF

5630

38OP

U_BS

_Ea2

012-

33

Ephe

mer

ella

ata

gosa

na

nym

phw

hole

bod

ySh

iga,

Japa

nM

ar, 2

012

KF56

2889

KF56

2984

KF56

3039

OPU_

BS_E

a201

2-34

Ephe

mer

ella

ata

gosa

na

nym

phhe

mol

ymph

Kana

gaw

a, Ja

pan

Mar

, 201

2KF

5628

92KF

5629

86KF

5630

52OP

U_BS

_Ea2

012-

43

Ephe

mer

ella

ata

gosa

na

nym

phhe

mol

ymph

Kana

gaw

a, Ja

pan

Mar

, 201

2KF

5628

93KF

5629

87OP

U_BS

_Ea2

012-

44

Ephe

mer

ella

ata

gosa

na

nym

phw

hole

bod

yKa

naga

wa,

Japa

nM

ar, 2

012

KF56

2988

OPU_

BS_E

a201

2-45

Ephe

mer

ella

not

ata

nym

phle

gKy

oto,

Japa

n11

Apr

, 201

4

KP

9707

22OP

U_BS

_En2

014-

17

Ephe

mer

ella

not

ata

nym

phle

gKy

oto,

Japa

n11

Apr

, 201

4KP

9707

98OP

U_BS

_En2

014-

18

Ephe

mer

ella

not

ata

nym

phle

gKy

oto,

Japa

n13

Apr

, 201

4KP

9707

99KP

9707

23OP

U_BS

_En2

014-

22

Ephe

mer

ella

not

ata

nym

phle

gKy

oto,

Japa

n13

Apr

, 201

4

KP

9707

24OP

U_BS

_En2

014-

23

Ephe

mer

ella

sp.

imag

osp

erm

vesi

cles

Nara

, Jap

an25

Apr

, 200

9GU

3541

55Ex

x200

9-16

-Oot

aki-D

Ephe

mer

ella

sp.

nym

phhe

mol

ymph

Kyus

hu, J

apan

Mar

, 201

2KF

5629

04KF

5629

91KF

5630

53OP

U_BS

_E20

12-8

6

Ephe

mer

ella

sp.

nym

phhe

mol

ymph

Kyus

hu, J

apan

Mar

, 201

2KF

5629

59KF

5630

54OP

U_BS

_E20

12-9

0

Ephe

mer

ella

sp.

nym

phhe

mol

ymph

Kyus

hu, J

apan

Mar

, 201

2KF

5629

05KF

5629

92OP

U_BS

_E20

12-9

1

Ephe

mer

ella

sp.

imag

ow

ing

mus

cle

Wak

ayam

a, Ja

pan

5 M

ay, 2

012

KF56

2963

KF56

3051

OPU_

BS_E

2012

-129

Ephe

mer

ella

sp.

imag

osp

erm

vesi

cles

Nara

, Jap

an27

Apr

, 201

3KP

9707

29OP

U_BS

_E20

13-5

1

Ephe

mer

ella

sp.

nym

phab

dom

enOs

aka,

Japa

n29

Jun,

201

4KP

9708

00KP

9707

25OP

U_BS

_E20

14-6

8

Ephe

mer

ella

sp.

nym

phab

dom

enOs

aka,

Japa

n29

Jun,

201

4KP

9708

01KP

9707

26OP

U_BS

_E20

14-6

9

Ephe

mer

ella

sp.

nym

phab

dom

enOs

aka,

Japa

n29

Jun,

201

4KP

9707

27OP

U_BS

_E20

14-7

1

Ephe

mer

ella

sp.

nym

phab

dom

enOs

aka,

Japa

n29

Jun,

201

4KP

9708

02OP

U_BS

_E20

14-7

2

Ephe

mer

ella

sp.

nym

phab

dom

enOs

aka,

Japa

n29

Jun,

201

4KP

9708

03KP

9707

28OP

U_BS

_E20

14-7

3

Ephe

mer

ella

sp.

nym

phab

dom

enOs

aka,

Japa

n29

Jun,

201

4KP

9708

04OP

U_BS

_E20

14-7

6

Serr

atel

la s

etig

era

nym

phw

hole

bod

yNa

ra, J

apan

15 M

ay, 2

011

JQ

6501

25

OPU_

BS_S

s201

1-16

7

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Serr

atel

la s

etig

era

nym

phw

hole

bod

yW

akay

ama,

Japa

n16

Nov

, 201

1JQ

6501

99JQ

6501

42JQ

6551

13OP

U_BS

_Es2

011-

605

Telo

gano

psis

bro

cha

nym

phw

hole

bod

yTa

iwan

Sep,

201

1KF

5628

73KF

5629

56JQ

6551

19OP

U_BS

_Tb2

011-

542

Telo

gano

psis

bro

cha

nym

phw

hole

bod

yTa

iwan

Sep,

201

1KF

5629

57OP

U_BS

_Tb2

011-

543

Torle

ya ja

poni

ca

nym

phhe

mol

ymph

Okin

awa,

Japa

n22

Apr

, 201

2KF

5629

25

OP

U_BS

_Tj2

012-

193

Torle

ya ja

poni

ca

nym

phhe

mol

ymph

Okin

awa,

Japa

n22

Apr

, 201

2KF

5629

26KF

5629

95

OPU_

BS_T

j201

2-19

4

Urac

anth

ella

chin

oiny

mph

leg

Okay

ama,

Japa

n16

Sep

, 201

2KP

9707

84KP

9707

38KP

9707

04OP

U_BS

_Uc2

013-

356

Urac

anth

ella

chin

oiny

mph

leg

Okay

ama,

Japa

n16

Sep

, 201

2KP

9707

85KP

9707

05OP

U_BS

_Uc2

013-

357

Urac

anth

ella

pun

ctis

etae

nym

phsp

erm

vesi

cles

Nara

, Jap

an25

Apr

, 200

9GU

3541

54GQ

4339

98

Uru2

009-

10-O

osak

o-D

Ephe

mer

ellid

ae s

p.su

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0777

OPU_

BS_E

2013

-167

Ephe

mer

ellid

ae s

p.su

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0778

OPU_

BS_E

2013

-168

Ephe

mer

ellid

ae s

p.su

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0779

OPU_

BS_E

2013

-169

Ephe

mer

ellid

ae s

p.su

bim

ago

leg

Wak

ayam

a, Ja

pan

11 M

ay, 2

014

KP97

0715

OPU_

BS_E

2014

-4

Baet

idae

Acen

trella

gno

mny

mph

thor

axKy

oto,

Japa

n20

Sep

, 201

0KP

9707

88KP

9707

09OP

U_BS

_Ag2

013-

361

Acen

trella

gno

mny

mph

abdo

men

Kyot

o, Ja

pan

20 S

ep, 2

010

KP97

0789

KP97

0739

KP97

0710

OPU_

BS_A

g201

3-36

2

Acen

trella

sibi

rica

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KF56

2899

KF56

2980

OP

U_BS

_As2

012-

254

Acen

trella

sibi

rica

subi

mag

oth

orax

Nara

, Jap

an27

Apr

, 201

3

KP

9707

01OP

U_BS

_As2

013-

145

Alai

nite

s yos

hine

nsis

nym

phw

hole

bod

yKa

naga

wa,

Japa

n9

Nov,

2011

KF56

2874

OPU_

BS_A

y201

1-56

6

Alai

nite

s yos

hine

nsis

subi

mag

ow

ing

mus

cle

Nara

, Jap

an27

Apr

, 201

3KP

9706

97OP

U_BS

_Ay2

013-

131

Alai

nite

s yos

hine

nsis

subi

mag

ow

ing

mus

cle

Nara

, Jap

an27

Apr

, 201

3KP

9706

98OP

U_BS

_Ay2

013-

132

Baet

iella

japo

nica

subi

mag

osp

erm

vesi

cles

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

JQ

6501

49

OPU_

BS_B

j201

0-38

Baet

iella

japo

nica

subi

mag

ow

ing

mus

cle

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

JQ65

0211

JQ65

0150

KF56

3015

OPU_

BS_B

j201

0-39

Baet

iella

japo

nica

subi

mag

osp

erm

vesi

cles

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

JQ65

0151

OPU_

BS_B

j201

0-40

Baet

iella

japo

nica

subi

mag

ow

ing

mus

cle

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

KF56

3016

OPU_

BS_B

j201

0-41

Baet

is th

erm

icus

nym

phw

hole

bod

yKa

naga

wa,

Japa

n9

Nov,

2011

KF56

2972

KF56

3030

OPU_

BS_B

t201

1-56

4

Baet

is th

erm

icus

nym

phw

hole

bod

yKa

naga

wa,

Japa

n16

Nov

, 201

1JQ

6502

00KF

5629

73KF

5630

31OP

U_BS

_Bt2

011-

606

Baet

is th

erm

icus

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KF56

2900

OPU_

BS_B

t201

2-26

1

Baet

is s

p.

imag

osp

erm

vesi

cles

Nara

, Jap

an25

Apr

, 200

9GU

3541

52

Bx

x200

9-07

-Oos

ako-

U

Baet

is s

p.im

ago

win

g m

uscl

eW

akay

ama,

Japa

n24

Apr

, 201

0JQ

6551

11OP

U_BS

_B20

10-2

3

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Baet

is s

p.

imag

ow

ing

mus

cle

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

JQ65

5112

OPU_

BS_B

2010

-23

clon

e 2

Baet

is s

p.im

ago

win

g m

uscl

eW

akay

ama,

Japa

n24

Apr

, 201

0JQ

6502

08OP

U_BS

_B20

10-2

4

Baet

is s

p.im

ago

win

g m

uscl

eNa

ra, J

apan

14 A

pr, 2

011

JQ65

0173

JQ65

0129

OPU_

BS_B

2011

-19

Baet

is s

p.im

ago

sper

m ve

sicl

esNa

ra, J

apan

14 A

pr, 2

011

JQ65

0182

JQ65

0158

OPU_

BS_B

2011

-74

Baet

is s

p.im

ago

win

g m

uscl

eNa

ra, J

apan

15 A

pr, 2

011

JQ65

0192

JQ65

0138

OPU_

BS_B

2011

-147

Baet

is s

p.im

ago

who

le b

ody

Nara

, Jap

an14

May

, 201

1KF

5629

50JQ

6551

16OP

U_BS

_B20

11-4

94

Baet

is s

p.su

bim

ago

thor

axNa

ra, J

apan

27 A

pr, 2

013

KP97

0694

OPU_

BS_B

2013

-85

Baet

is s

p.su

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0695

OPU_

BS_B

2013

-101

Baet

is s

p.im

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0770

OPU_

BS_B

2013

-102

Baet

is s

p.su

bim

ago

sper

m ve

sicl

esNa

ra, J

apan

27 A

pr, 2

013

KP97

0771

OPU_

BS_B

2013

-103

Baet

is s

p.su

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0699

OPU_

BS_B

2013

-134

Baet

is s

p.im

ago

thor

axNa

ra, J

apan

27 A

pr, 2

013

KP97

0776

OPU_

BS_B

2013

-166

Labi

obae

tis a

treba

tinus

or

ient

alis

nym

phw

hole

bod

yKa

naga

wa,

Japa

n24

Nov,

2011

KF56

2878

KF56

3032

OPU_

BS_L

a201

1-61

4

Labi

obae

tis tr

icol

orny

mph

thor

axCh

iba,

Japa

n9

May

, 200

9

KP97

0734

OP

U_BS

_Lt2

013-

352

Labi

obae

tis tr

icol

orny

mph

thor

axCh

iba,

Japa

n9

May

, 200

9KP

9707

82KP

9707

35

OPU_

BS_L

t201

3-35

3

Nigr

obae

tis ch

ocor

atus

nym

phw

hole

bod

yW

akay

ama,

Japa

n5

May

, 201

2KF

5629

40KF

5629

75KF

5630

60OP

U_BS

_Nc2

012-

227

Nigr

obae

tis s

p. D

nym

phw

hole

bod

yW

akay

ama,

Japa

n5

May

, 201

2KF

5629

39KF

5630

02KF

5630

59OP

U_BS

_N20

12-2

26

Nigr

obae

tis ta

iwan

ensi

s ny

mph

who

le b

ody

Kana

gaw

a, Ja

pan

25 N

ov, 2

011

KF56

2884

OPU_

BS_B

t201

1-60

7

Nigr

obae

tis ta

iwan

ensi

sny

mph

who

le b

ody

Kana

gaw

a, Ja

pan

25 N

ov, 2

011

KF56

2883

OPU_

BS_B

t201

1-60

8

Tenu

ibae

tis fl

exife

mor

any

mph

thor

axKy

oto,

Japa

n20

Sep

, 201

0KP

9707

90KP

9707

40KP

9707

11OP

U_BS

_Tf2

013-

363

Tenu

ibae

tis fl

exife

mor

any

mph

thor

axKy

oto,

Japa

n20

Sep

, 201

0KP

9707

91KP

9707

41KP

9707

12OP

U_BS

_Tf2

013-

364

Baet

idae

sp.

imag

ole

gW

akay

ama,

Japa

n11

May

, 201

4KP

9707

16OP

U_BS

_B20

14-5

Baet

idae

sp.

imag

ole

gW

akay

ama,

Japa

n11

May

, 201

4KP

9707

17OP

U_BS

_B20

14-6

Ephe

mer

idae

Ephe

mer

a fo

rmos

ana

imag

ow

ing

mus

cle

Taiw

anSe

p, 2

011

JQ65

0223

OPU_

BS_E

f201

1-52

5

Ephe

mer

a fo

rmos

ana

imag

osp

erm

vesi

cles

Taiw

anSe

p, 2

011

JQ65

0224

JQ65

0164

JQ65

5117

OPU_

BS_E

f201

1-52

6

Ephe

mer

a ja

poni

cany

mph

who

le b

ody

Miy

agi,

Japa

n8

Aug,

201

0

JQ65

0167

OP

U_BS

_Ej2

010-

EJ1

Ephe

mer

a ja

poni

cany

mph

who

le b

ody

Miy

agi,

Japa

n8

Aug,

201

0JQ

6501

68OP

U_BS

_Ej2

010-

EJ2

Ephe

mer

a ja

poni

cany

mph

who

le b

ody

Miy

agi,

Japa

n8

Aug,

201

0JQ

6501

69OP

U_BS

_Ej2

010-

EJ3

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Ephe

mer

a ja

poni

cany

mph

who

le b

ody

Kana

gaw

a, Ja

pan

12 S

ep, 2

011

JQ65

0153

OPU_

BS_E

j201

1-50

7

Ephe

mer

a ja

poni

cany

mph

hem

olym

phKa

naga

wa,

Japa

n12

Sep

, 201

1JQ

6501

97OP

U_BS

_Ej2

011-

508

Ephe

mer

a ja

poni

cany

mph

win

g m

uscl

eFu

kush

ima,

Japa

n11

Sep

, 201

1KF

5629

43OP

U_BS

_Ej2

012-

265

Ephe

mer

a ja

poni

cany

mph

win

g m

uscl

eFu

kush

ima,

Japa

n11

Sep

, 201

1KF

5629

44OP

U_BS

_Ej2

012-

266

Ephe

mer

a ja

poni

cany

mph

win

g m

uscl

eFu

kush

ima,

Japa

n11

Sep

, 201

1KF

5629

45OP

U_BS

_Ej2

012-

267

Ephe

mer

a ja

poni

cany

mph

win

g m

uscl

eFu

kush

ima,

Japa

n11

Sep

, 201

1KF

5629

46OP

U_BS

_Ej2

012-

268

Ephe

mer

a ja

poni

cany

mph

win

g m

uscl

eFu

kush

ima,

Japa

n11

Sep

, 201

1KF

5629

47OP

U_BS

_Ej2

012-

269

Ephe

mer

a ja

poni

cany

mph

win

g m

uscl

eFu

kush

ima,

Japa

n11

Sep

, 201

1KF

5629

48

OP

U_BS

_Ej2

012-

270

Ephe

mer

a or

ient

alis

nym

phw

ing

mus

cle

Shig

a, Ja

pan

Mar

, 201

2KF

5628

90KF

5629

85OP

U_BS

_Eo2

012-

35

Ephe

mer

a or

ient

alis

nym

phw

ing

mus

cle

Shig

a, Ja

pan

Mar

, 201

2KF

5628

91KF

5629

58OP

U_BS

_Eo2

012-

36

Ephe

mer

a st

rigat

aim

ago

sper

m ve

sicl

esKy

oto,

Japa

n

EF68

0328

ES20

02Ky

oto1

Ephe

mer

a st

rigat

aim

ago

sper

m ve

sicl

esKy

oto,

Japa

nEF

6803

29ES

2002

Kyot

o2

Ephe

mer

a st

rigat

aim

ago

sper

m ve

sicl

esKy

oto,

Japa

nAY

8608

41ES

2002

Kyot

o

Ephe

mer

a st

rigat

aim

ago

sper

m ve

sicl

esNa

ra, J

apan

25 A

pr, 2

009

GU35

4151

Est2

009-

06-O

osak

o-U

Ephe

mer

a st

rigat

any

mph

who

le b

ody

Miy

agi,

Japa

n8

Aug,

201

0JQ

6501

70OP

U_BS

_Es2

010-

ES2

Ep

hem

era

strig

ata

nym

phw

hole

bod

yM

iyag

i, Ja

pan

8 Au

g, 2

010

JQ

6501

71JQ

6551

14OP

U_BS

_Es2

010-

ES3

Lept

ophl

ebiid

ae Chor

oter

pide

s ni

gella

nym

phw

hole

bod

yTa

iwan

Sep,

201

1JQ

6502

25JQ

6501

65JQ

6551

18OP

U_BS

_Cn2

011-

534

Chor

oter

pide

s ni

gella

nym

phw

hole

bod

yTa

iwan

Sep,

201

1JQ

6501

66OP

U_BS

_Cn2

011-

539

Para

lept

ophl

ebia

choc

olat

aim

ago

sper

m ve

sicl

esNa

ra, J

apan

25 A

pr, 2

009

GQ

4339

99GU

3541

61Pc

h200

9-17

-Oot

aki-D

Para

lept

ophl

ebia

choc

olat

aim

ago

sper

m ve

sicl

esNa

ra, J

apan

25 A

pr, 2

009

GQ

4340

00

Pch2

009-

18-O

otak

i-D

Para

lept

ophl

ebia

japo

nica

subi

mag

osp

erm

vesi

cles

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

JQ65

0204

JQ65

0144

OPU_

BS_P

j201

0-13

Para

lept

ophl

ebia

japo

nica

subi

mag

ow

ing

mus

cle

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

JQ65

0205

JQ65

0145

KF56

3011

OPU_

BS_P

j201

0-14

Para

lept

ophl

ebia

spin

osa

imag

osp

erm

vesi

cles

Wak

ayam

a, Ja

pan

9 Ap

r, 20

06GU

3541

46

PS

2006

Niug

awa-

1

Para

lept

ophl

ebia

spin

osa

imag

osp

erm

vesi

cles

Wak

ayam

a, Ja

pan

9 Ap

r, 20

06EF

0554

51PS

2006

Niug

awa

1-2-

3

Para

lept

ophl

ebia

spin

osa

imag

osp

erm

vesi

cles

Nara

, Jap

an14

Apr

, 201

1JQ

6501

80JQ

6501

34OP

U_BS

_Ps2

011-

41

Para

lept

ophl

ebia

spin

osa

imag

osp

erm

vesi

cles

Nara

, Jap

an14

Apr

, 201

1JQ

6501

81JQ

6501

35

OPU_

BS_P

s201

1-42

Para

lept

ophl

ebia

wes

toni

nym

phw

hole

bod

yKa

naga

wa,

Japa

n12

Sep

, 201

1KF

5629

51OP

U_BS

_Pw

2011

-495

Para

lept

ophl

ebia

wes

toni

nym

phw

hole

bod

yKa

naga

wa,

Japa

n12

Sep

, 201

1KF

5629

52OP

U_BS

_Pw

2011

-496

Para

lept

ophl

ebia

wes

toni

nym

phw

hole

bod

yKa

naga

wa,

Japa

n12

Sep

, 201

1JQ

6502

19OP

U_BS

_Pw

2011

-502

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Para

lept

ophl

ebia

wes

toni

imag

ow

ing

mus

cle

Kana

gaw

a, Ja

pan

12 S

ep, 2

011

JQ65

0196

KF56

3025

OPU_

BS_P

w20

11-5

04

Para

lept

ophl

ebia

sp.

subi

mag

ow

ing

mus

cle

Nara

, Jap

an8

May

, 201

0JQ

6502

09JQ

6501

48KF

5630

18OP

U_BS

_P20

10-3

62

Para

lept

ophl

ebia

sp.

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KF56

2978

OPU_

BS_P

2012

-248

Para

lept

ophl

ebia

sp.

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KF56

2979

OPU_

BS_P

2012

-249

Para

lept

ophl

ebia

sp.

imag

ow

ing

mus

cle

Nara

, Jap

an27

Apr

, 201

3KP

9707

72OP

U_BS

_P20

13-1

05

Para

lept

ophl

ebia

sp.

subi

mag

ow

ing

mus

cle

Nara

, Jap

an27

Apr

, 201

3KP

9707

73KP

9707

31OP

U_BS

_P20

13-1

06

Para

lept

ophl

ebia

sp.

imag

osp

erm

vesi

cles

Wak

ayam

a, Ja

pan

28 A

pr, 2

013

KP97

0732

OPU_

BS_P

2013

-200

Para

lept

ophl

ebia

sp.

subi

mag

osp

erm

vesi

cles

Wak

ayam

a, Ja

pan

28 A

pr, 2

013

KP97

0780

KP97

0733

OP

U_BS

_P20

13-2

02

Thra

ulus

gra

ndis

nym

phhe

mol

ymph

Okin

awa,

Japa

n23

Apr

, 201

2KF

5629

30KF

5629

96OP

U_BS

_Tg2

012-

210

Thra

ulus

gra

ndis

nym

phhe

mol

ymph

Okin

awa,

Japa

n23

Apr

, 201

2KF

5629

31KF

5629

97KF

5630

57OP

U_BS

_Tg2

012-

211

Thra

ulus

gra

ndis

nym

phhe

mol

ymph

Okin

awa,

Japa

n23

Apr

, 201

2KF

5629

32KF

5629

98OP

U_BS

_Tg2

012-

212

Thra

ulus

gra

ndis

nym

phw

hole

bod

yOk

inaw

a, Ja

pan

23 A

pr, 2

012

KF56

2933

KF56

2999

OP

U_BS

_Tg2

012-

214

Amel

etid

ae

Amel

etus

cost

alis

imag

osp

erm

vesi

cles

Kyot

o, Ja

pan

Apr,

1997

EF68

0323

AY87

0291

AC19

97Ki

bune

-O

Amel

etus

cost

alis

imag

ow

ing

mus

cle

Nara

, Jap

an14

Apr

, 201

1JQ

6501

79JQ

6501

33KF

5630

21OP

U_BS

_Ac2

011-

38

Amel

etus

cost

alis

imag

ow

ing

mus

cle

Nara

, Jap

an15

Apr

, 201

1JQ

6501

88JQ

6501

60OP

U_BS

_Ac2

011-

108

Amel

etus

mon

tanu

sim

ago

sper

m ve

sicl

esNa

ra, J

apan

25 A

pr, 2

009

GQ

4339

95

Amo2

009-

04-

Oosa

ko-U

Amel

etus

mon

tanu

sim

ago

ovar

yNa

ra, J

apan

25 A

pr, 2

009

GU35

4150

GQ43

3996

Amo2

009-

05-

Oosa

ko-U

Amel

etus

mon

tanu

ssu

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0765

KP97

0730

KP97

0688

OPU_

BS_A

m20

13-6

7

Amel

etus

mon

tanu

ssu

bim

ago

win

g m

uscl

eNa

ra, J

apan

27 A

pr, 2

013

KP97

0766

KP

9706

89OP

U_BS

_Am

2013

-68

Amel

etus

sp.

imag

osp

erm

vesi

cles

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

JQ65

0215

JQ65

0152

OPU_

BS_A

2010

-9

Amel

etus

sp.

imag

ow

ing

mus

cle

Wak

ayam

a, Ja

pan

24 A

pr, 2

010

JQ65

0201

JQ65

0143

KF56

3009

OPU_

BS_A

2010

-10

Amel

etus

sp.

imag

ow

ing

mus

cle

Nara

, Jap

an15

Apr

, 201

1JQ

6501

87JQ

6501

37OP

U_BS

_A20

11-1

06

Amel

etus

sp.

subi

mag

ow

ing

mus

cle

Nara

, Jap

an15

Apr

, 201

0JQ

6501

90JQ

6501

22OP

U_BS

_A20

11-1

22

Amel

etus

sp.

nym

phw

hole

bod

yNa

ra, J

apan

14 D

ec, 2

012

KF56

2901

KF56

2981

OPU_

BS_A

2012

-262

Am

elet

us s

p.ny

mph

leg

Kyot

o, Ja

pan

11 A

pr, 2

014

KP

9707

47

OPU_

BS_A

2014

-12

Pota

man

thid

ae

Pota

man

thus

form

osus

nym

phhe

mol

ymph

Fuku

oka,

Japa

n15

Apr

, 201

2KF

5629

24KF

5629

94KF

5630

56OP

U_BS

_Pf2

012-

192

Pota

man

thus

form

osus

nym

phle

gKy

oto,

Japa

n11

Apr

, 201

4KP

9707

95KP

9707

48OP

U_BS

_Pf2

014-

13

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


Fam

ily n

ame

Spec

ies

nam

eSt

age

Sour

ce ti

ssue

Loca

tion

Colle

ctio

n Da

te18

S rR

NAHi

ston

e H3

COI

Spe

cim

en vo

uche

r

Pota

man

thus

form

osus

imag

ole

gNa

ra, J

apan

6 Ju

l, 20

14

KP97

0749

OP

U_BS

_Pf2

014-

77

Pota

man

thus

idio

ceru

sim

ago

sper

m ve

sicl

esTa

iwan

Sep,

201

1JQ

6502

22KF

5629

54OP

U_BS

_Pi2

011-

521

Po

tam

anth

us id

ioce

rus

imag

ow

ing

mus

cle

Taiw

anSe

p, 2

011

KF

5629

55KF

5630

29OP

U_BS

_Pi2

011-

522

Siph

lonu

ridae

Siph

lonu

rus s

anuk

ensi

sny

mph

win

g m

uscl

eFu

kuok

a, Ja

pan

16 A

pr, 2

012

KF56

2915

KF56

2970

OPU_

BS_S

s201

2-17

4

Siph

lonu

rus s

anuk

ensi

sny

mph

hem

olym

phFu

kuok

a, Ja

pan

16 A

pr, 2

012

KF56

2916

KF56

2993

OPU_

BS_S

s201

2-17

5

Siph

lonu

rus s

anuk

ensi

sny

mph

hem

olym

phFu

kuok

a, Ja

pan

16 A

pr, 2

012

KF

5629

71KF

5630

47OP

U_BS

_Ss2

012-

176

Siph

lonu

rus

sp.

nym

phle

gKy

oto,

Japa

n11

Apr

, 201

4KP

9707

92KP

9707

42KP

9707

18OP

U_BS

_S20

14-7

Siph

lonu

rus

sp.

nym

phle

gKy

oto,

Japa

n11

Apr

, 201

4KP

9707

93KP

9707

43KP

9707

19OP

U_BS

_S20

14-8

Siph

lonu

rus

sp.

nym

phle

gKy

oto,

Japa

n11

Apr

, 201

4KP

9707

94KP

9707

44KP

9707

20OP

U_BS

_S20

14-9

Siph

lonu

rus

sp.

nym

phle

gKy

oto,

Japa

n11

Apr

, 201

4KP

9707

45KP

9707

21OP

U_BS

_S20

14-1

0

Si

phlo

nuru

s sp

.ny

mph

leg

Kyot

o, Ja

pan

11 A

pr, 2

014

KP

9707

46

OPU_

BS_S

2014

-11

Caen

idae

Ca

enis

sp.

imag

ow

hole

bod

yNa

ra, J

apan

14 M

ay, 2

011

KP97

0805

KP97

0757

OP

U_BS

_C20

11-4

93

Ison

ychi

idae

Is

onyc

hia

japo

nica

nym

phw

ing

mus

cle

Wak

ayam

a, Ja

pan

15 M

ay, 2

011

KF56

2949

KF56

3006

OP

U_BS

_Ij2

011-

169

Poly

mita

rcyi

dae Ep

horo

n sh

igae

nym

phth

orax

Okay

ama,

Japa

n16

Sep

, 201

1KP

9707

36KP

9707

02OP

U_BS

_Es2

013-

354

Ep

horo

n sh

igae

nym

phth

orax

Okay

ama,

Japa

n16

Sep

, 201

1KP

9707

83KP

9707

37KP

9707

03OP

U_BS

_Es2

013-

355

Tabl

e 1.

Acc

essi

on n

umbe

rs o

f seq

uenc

e da

ta a

nd s

ourc

e in

form

atio

n.


3 Results and discussionA total of 486 sequences data (118 of COI, 214 of 18S rRNA, and 154 of histone H3) have been deposited in GenBank/ EMBL/ DDBJ International Nucleotide Databases, from which they can be retrieved using the search terms “Ephemeroptera” + “Osaka Prefecture” + gene name. Accession numbers of sequence data are listed on Table 1. Lack of an accession number means the sequence was not determined. Some of them were difficult to amplify by PCR under the conditions indicated, and some were postponed because of lower priority of analysis (large effort was made to analyze the unsequenced species). A portion of the DNA sequences (98 among 486 sequences) were not registered with full scientific names due to our conservative way of identification, thus were not applicable to DNA barcoding at the species level. However, their registration with precise information on sampling locations and time (year/month) is still beneficial for ecological and conservation studies. Comparing with sequence data from undoubtedly identified specimens, which was already registered in the database or will be registered in the course of future studies, the sequences from our ambiguous specimens may be used in the estimation of habitat range and its temporal change.

In the phylogenetic trees of COI and H3, the family Heptageniidae was divided into three distant clusters or singletons (Figs. 1A and 1B). This could be the result of long branch attraction [14] due to the highly variable nature of these genes, or saturation of substitutions at the third positions of codons [15]. The low levels of support for the estimated phylogenetic relationships at higher taxonomic levels (i.e., family and genus), however, does not lessen the validity of species-level identification [16,17]. The 18S rRNA tree showed monophyletic clustering of families (Fig. 1C) when the highly variable V4 stem-loop regions (containing many insertions/deletions that were impossible to align unambiguously) were excluded from the analysis.

The specimen of Nigrobaetis sp. D (N2012-226) was recovered in its expected position as sister-group to Nigrobaetis chocoratus (Nc2012-227) in both the COI and 18S trees (Fig. 1A and Fig. 1C) but in the histone H3 tree (Fig. 1B) it is placed well within a clade comprising Empherellidae specimens, as sister-group to Ephemeralla sp. (E2012-86). It is possible that we amplified a paralogous copy of the histone H3 gene in this instance, or that there might have been cross-contamination of samples, however as we do not have any further specimens of Nigrobaetis sp. D we cannot test this hypothesis.

Our results suggest that because of their higher evolutionary rates, COI and H3 are not suitable in inferring phylogenetic relationship among families but were useful in identifying species (i.e., DNA barcoding). In contrast, the more conserved 18S rRNA gene was useful in confirming the integrity of the source DNA samples (e.g. contamination) and was adequately informative for elucidating inter-family divergence, but not suitable for species identification. We also found intraspecific variations in COI sequences of some species (Fig. 2), that can be applicable to characterizing the genetic structures of populations. The maximum intraspecific variation seen in Ephemerella atagosana and Drunella basalis was 4.9% and 3.3%, respectively. This may suggest that cryptic species exist within these species.

In conclusion, we collected DNA samples of 80 taxa of Ephemeroptera, covering ca. 60% of mayfly species in Japan. We analyzed COI, histone H3, and 18S rRNA sequences with 39, 46, and 61 species, respectively, which were morphologically identified. Our results indicated that COI, histone H3 gene sequences could serve as markers for species identification, whereas more conserved 18S rRNA gene could serve a marker for quality control of DNA samples. Sequence data presented in this study would also contribute to the population genetics studies by comparing the haplotype frequency of certain species in the future.

Table 2. DNA primers used for PCR and sequencing analysis.

Primer name Sequence Target Annealing temperature Reference

HexAF atggctcgtaccaagcagacggc histone H3 55°C Ogden & Whiting [18]

HexAR atatccttgggcatgatggtgac

LCO1490 ggtcaacaaatcataaagatattgg cytochrome oxidase subunit 1 45°C Folmer et al. [19]

HCO2198 taaacttcagggtgaccaaaaaatca

KOBO18SF1 tggcgtatattaaagttgttgcggt V4 and V5 region of 18S rRNA 55°C Kobori et al., unpublished.

KOBO18SR1 agtttcagctttgcaaccatacttc


Figure 1A. Phylogenetic trees of three genes reconstructed with nucleotide sequences. A, COI tree; B, histone H3 tree; C, 18S rRNA tree.Phylogenetic trees were reconstructed by the neighbor-joining method with sequence data of three genes (COI, H3, and 18S rRNA). The trees were rooted using outgroup sequences from Odonata. Species names were followed by the specimen IDs. Bootstrap reproducibility scores (≥50 %) were indicated at respective nodes. Specimen IDs marked with asterisk (*) indicates that a whole body was used for DNA extrac-tion (i.e., unable to re-examine species morphologically). The V4 stem-loop region of 18S rRNA (corresponding to the position 650-788 of X89481, Misof et al. [5]) was trimmed from the sequence data in this phylogenetic analysis.


Figure 1B. Phylogenetic trees of three genes reconstructed with nucleotide sequences. A, COI tree; B, histone H3 tree; C, 18S rRNA tree.Phylogenetic trees were reconstructed by the neighbor-joining method with sequence data of three genes (COI, H3, and 18S rRNA). The trees were rooted using outgroup sequences from Odonata. Species names were followed by the specimen IDs. Bootstrap reproducibility scores (≥50 %) were indicated at respective nodes. Specimen IDs marked with asterisk (*) indicates that a whole body was used for DNA extrac-tion (i.e., unable to re-examine species morphologically). The V4 stem-loop region of 18S rRNA (corresponding to the position 650-788 of X89481, Misof et al. [5]) was trimmed from the sequence data in this phylogenetic analysis.


Figure 1C. Phylogenetic trees of three genes reconstructed with nucleotide sequences. A, COI tree; B, histone H3 tree; C, 18S rRNA tree.Phylogenetic trees were reconstructed by the neighbor-joining method with sequence data of three genes (COI, H3, and 18S rRNA). The trees were rooted using outgroup sequences from Odonata. Species names were followed by the specimen IDs. Bootstrap reproducibility scores (≥50 %) were indicated at respective nodes. Specimen IDs marked with asterisk (*) indicates that a whole body was used for DNA extrac-tion (i.e., unable to re-examine species morphologically). The V4 stem-loop region of 18S rRNA (corresponding to the position 650-788 of X89481, Misof et al. [5]) was trimmed from the sequence data in this phylogenetic analysis.


help and support in collecting mayfly samples in Kii-Nyugawa River. This work was partially supported by Water Resources Environment Technology Center (Grant Number 2008-04) and Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT KAKENHI Grant Number 25289172).

Acknowledgements: The authors would like to thank Ms. Tamami Adachi for her help in DNA sequencing, Dr. Toshihito Fujitani for identification of some specimens belonging to Baetidae, and Tamagawakyo-wo-Mamorukai (Hashimoto City, Wakayama, Japan), a citizens’ group working for nature studies and conservation, for their

Figure 2. Alignment of COI sequences with intraspecific variations.Sequences are labeled with specimen IDs, and the species for each specimen ID are as follows: Drunella basalis, Db2012-1, Db2012-2, Db2012-5, Db2012-6, Db2012-7, Db2012-100, Db2012-101, Db2012-160, Db2012-161, and Db2012-164; Rhithrogena japonica, Rj2008-64, Rj2008-65, Rj2008-68, Rj2008-69, and Rj2005-10; Ephemerella atagosana, Ea2012-33, Ea2012-34, and Ea2012-43; Ephemerella notate, En2014-17, En2014-22, and En2014-23; Rhithrogena tetrapunctigera, Rt2013-358 and Rt2013-359; Drunella sachalinensis, Ds2012-54 and 2012-55; Uracanthella chinoi, Uc2013-356 and Uc2013-357; Acentrella gnom, Ag2013-361 and 2013-362; Ameletus montanus, Am2013-67 and Am2013-68; Baetiella japonica, Bj2010-39 and Bj2010-41; Ephoron shigae, Es2013-354 and Es2013-355.


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Supplemental Material: The online version of this article(DOI: 10.1515/dna-2016-0001) offers supplementary material.

Conflict of interest: Authors declare nothing to disclose.

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kazumi tanida , nobuyoshi shimizu, mikio kato

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