

DNA Barcodes 2016; 4: 1–19

Research Article

Open Access

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

Abstract: We have determined nucleotide sequencesof three genes encoding histone H3, 18S rRNA, andmitochondrial cytochrome oxidase subunit I (COI) fromthe 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 projectsof mayflies. Phylogenetic analysis based on the neighborjoining method among the three genes found that COI andH3 genes had relatively high evolutionary rates, and were
not suitable in inferring phylogenetic relationships among
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, Facultyof Liberal Arts and Sciences, Osaka Prefecture University,
1-1 Gakuencho, Naka-ku, Sakai 599-8531, Japan, E-mail:mkato@b.s.osakafu-u.ac.jp
Kei Wakimura, Biology Laboratory, Faculty of Liberal Arts andSciences, Osaka Prefecture University, 1-1 Gakuencho, Naka-ku,
Sakai 599-8531, Japan
Yasuhiro Takemon, Disaster Prevention Research Institute, Kyoto
University, Uji 611-0011, Japan
Atsushi Takayanagi, Nobuyoshi Shimizu, Advance Research Center
for Genome Super Power, Keio University, 2 Ohkubo, Tsukuba 3002611, Japan
Shin-ichi Ishiwata, Department of Applied Chemistry, Kanagawa
Institute of Technology, 1030 Shimo-Ogino, Atsugi 243-0292, Japan
Kozo Watanabe, Department of Civil and Environmental Engineering,
Ehime University, Bunkyo-cho 3, Matsuyama 790-8577, Japan
Kazumi Tanida, Mikio Kato, Department of Biological Sciences,
Graduate School of Science, Osaka Prefecture University, 1-1 Gakuencho, Naka-ku, Sakai 599-8531, Japan#
Present address: Osaka Museum of Natural History, Nagai Park,
Higashi-sumiyoshi, Osaka, 546-0034 Japanfamilies within the orders but were useful in identifying
species (i.e., DNA barcoding). In contrast, the moreconserved 18S rRNA gene was adequately informative forelucidating inter-family divergence, but not suitable for
species identification.
Keywords: COI; gene phylogeny; histone; mayfly; rRNA;
species identification

1 Introduction
Aquatic 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 ecologicalassessments, 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 whenhandling immature stages or damaged specimens. Asan alternative to the classical identification based onmorphological characters, DNA-based identification
had been proposed [5] and DNA barcoding (originallyproposed by Hebert et al. [6]) has become standard forcharacterizing a variety of taxonomic groups including
mayflies. DNA barcoding uses a matching analysis of ashort genetic marker (DNA barcode) to DNA databases ofidentified specimens. The utility and reliability of DNA
barcoding depend upon the quality of DNA databases inwhich the annotated sequence data are registered. While142 species of Japanese mayflies have been described from
39 genera in 13 families [7], only 17 species in 8 families of© 2016 Kei Wakimura, et al., published by De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.

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 insurveying 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 thephylogenetic 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 ofgenetic 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), and3 families (unidentified genus name) for sequencing
analysis (Table 1). In total, there were 33 genera
belonging to 11 families. These samples were collectedfrom Honshu, Kyushu and Ryukyu Islands of Japanand Taiwan in the years of 1997-2014. Specimens wereidentified by the authors before DNA extraction. To avoid
false-positive identification in DNA barcoding, only
accurately identified specimens must be registered withfull 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 absoluteethanol 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 thespecimens, followed by a conventional proteinase-phenol
extraction protocol [10]. Genitalia and heads parts were
kept intact during dissection as voucher specimens, andstored in absolute ethanol. However, the genitalium ofsome small specimens was damaged during the isolationof 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, bywhom 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 aresafely kept in the corresponding author’s laboratory(Osaka Prefecture University) except those safely storedin 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 bothforward and reverse directions using BigDye® Terminatorv3.1 (Life Technologies) and an ABI3730xl DNA Analyzer(Applied Biosystems - Life Technologies). Forward andreverse 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 werereconstructed based on the neighbor-joining method[12] using “Dnadist” with the option F84 model and“Neighbor” implemented in PHYLIP [13] with outgroupsequences (accession numbers: KC135891 for COI,
AY870292 for histone H3, EF680326 for 18S rRNA). Tree
topologies were examined by bootstrapping sequences of1000 pseudoreplicates using “Seqboot” in PHYLIP. For thetree reconstructions, we used only sequence data that has
a complete set of the three genes for simple comparisonof tree topologies among different genes. Incomplete data
sets (i.e., specimens of which only one or two genes outof three were determined) were not incorporated to theanalysis.

Heptageniidae
imago
imago
imago
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
imago
imago
imago
nymph
imago
imago
imago
nymph
nymph
imago
imago
imago
imago
nymph
nymph
nymph
nymph

Afronurus hyalinus

Afronurus hyalinus

Afronurus yoshidae

Afronurus yoshidae

Afronurus yoshidae

Afronurus yoshidae

Afronurus yoshidae

Bleptus fasciatus

Cinygmula grandifolia

Cinygmula grandifolia

Cinygmula sp.

Cinygmula sp.

Cinygmula sp.

Cinygmula sp.

Cinygmula sp.

Cinygmula sp.

Ecdyonurus bajkovae

Ecdyonurus bajkovae

Ecdyonurus bajkovae

Ecdyonurus tigris

Ecdyonurus tigris

Ecdyonurus tobiironis

Ecdyonurus tobiironis

Ecdyonurus viridis

Ecdyonurus viridis

Ecdyonurus viridis

Ecdyonurus sp.

Stage

Afronurus hyalinus

Family name Species name

Wakayama, Japan

Okinawa, Japan

Okinawa, Japan

Okinawa, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Wakayama, Japan

TaiwanTaiwanTaiwanLocation

15 May, 201123 Apr, 2012

23 Apr, 2012

23 Apr, 201227 Apr, 201327 Apr, 2013

8 May, 2010

8 May, 2010

5 May, 2012

5 May, 201226 Apr, 200926 Apr, 200926 Apr, 2009

5 May, 2012

5 May, 201214 Apr, 201114 Apr, 201123 Feb, 2014

23 Feb, 201417 Feb, 2005

25 Nov, 2011

25 Nov, 2011

25 Nov, 2011

25 Nov, 2011

15 May, 201127 Sep, 201127 Sep, 201127 Sep, 2011

Collection Date

KP970811

KF562929

KF562928

KF562927

KP970775

KP970774

JQ650206

KF562942

KF562941

GU354158

GU354157

GU354156

KF562938

KF562908

JQ650184

JQ650183

KP970797

KP970796

KF562882

KF562880

KF562881

KF562879

KF562867

JQ650221

JQ650220

18S rRNAJQ650162

JQ650147

KF563004

KF563003

KF563001

JQ650120

JQ650159

KF562953

Histone H3

GU354162

KF563024

KF563023

KP970708

KF563061

KF563028

KF563027

COI

OPU_BS_Ey2011-164

OPU_BS_Ev2012-199

OPU_BS_Ev2012-198

OPU_BS_Ev2012-197

OPU_BS_Et2013-150

OPU_BS_Et2013-148

OPU_BS_Et2010-214

OPU_BS_Et2010-212

OPU_BS_Eb2012-230

XXX2009-27Oosako-U
OPU_BS_Eb2012-229

Cxx2009-21-Niugawa

Cxx2009-20-Niugawa

OPU_BS_C2012-221

OPU_BS_C2012-130

OPU_BS_C2011-91

OPU_BS_C2011-90

OPU_BS_Cg2014-15

OPU_BS_Cg2014-14

OPU_BS_Bf2013-360

OPU_BS_Ey2011-628

OPU_BS_Ey2011-627

OPU_BS_Ey2011-626

OPU_BS_Ey2011-625

OPU_BS_Ey2011-168

OPU_BS_Eh2011-518

OPU_BS_Eh2011-517

OPU_BS_Eh2011-516

Specimen voucher

DNA barcodes of Japanese mayflies

whole body

hemolymph

hemolymph

hemolymph

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

whole bodywhole bodysperm vesicles

sperm vesicles

sperm vesicles

wing muscle

wing muscle

wing muscle

wing muscle

leg

leg

leg

leg

hemolymphleghemolymph

hemolymph

wing muscle

wing muscle

sperm vesicles

Source tissue

Table 1. Accession numbers of sequence data and source information.



3

nymph
nymph
imago
imago
imago
nymph
subimago
subimago
subimago
subimago
imago
nymph
nymph
imago
imago
imago
subimago
subimago
imago
imago
imago
imago
subimago
subimago
subimago
imago
imago
imago
imago
imago

Ecdyonurus sp.

Ecdyonurus sp.

Ecdyonurus sp.

Ecdyonurus sp.

Ecdyonurus sp.

Ecdyonurus sp.

Ecdyonurus sp.

Ecdyonurus sp.

Ecdyonurus sp.

Ecdyonurus sp.

Electrogena sp. 1

Electrogena sp. 1

Electrogena sp. 1

Epeorus aesculus

Epeorus aesculus

Epeorus aesculus

Epeorus curvatulus

Epeorus ikanonis

Epeorus ikanonis

Epeorus ikanonis

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifoliumStage

Ecdyonurus sp.

Family name Species namesperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

wing muscle

sperm vesicles

wing muscle

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

wing muscle

sperm vesicles

wing muscle

sperm vesicles

sperm vesicles

leg

leg

wing muscle

sperm vesicles

sperm vesicles

wing muscle

wing muscle

whole bodywing muscle

wing muscle

wing muscle

hemolymph

hemolymph

Source tissue

Table 1. Accession numbers of sequence data and source information.

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

Kyoto, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Fukuoka, Japan

Fukuoka, Japan

Location

14 Apr, 201114 Apr, 201114 Apr, 201114 Apr, 201114 Apr, 201125 Apr, 2010

25 Apr, 2010

25 Apr, 2010

25 Apr, 2004

14 Apr, 201114 Apr, 2011

Apr, 1997

8 May, 201027 Apr, 201327 Apr, 201327 Apr, 201312 Sep, 201112 Sep, 201112 Sep, 201127 Apr, 201327 Apr, 201327 Apr, 201327 Apr, 201327 Apr, 2013

5 May, 2012

5 May, 2012

5 May, 2012

5 May, 2012

Apr, 2012

Apr, 2012

Collection Date

JQ650177

KF562869

JQ650176

JQ650175

JQ650174

JQ650214

JQ650213

JQ650212

EF680333

JQ650172

EF680324

JQ650207

KP970764

KP970763

KP970762

JQ650217

JQ650216

KP970769

KP970768

KP970767

KP970810

KF562876

KF562910

KF562909

KF562923

KF562922

18S rRNAJQ650131

JQ650130

JQ650119

AY870290

JQ650118

JQ650128

AY870289

JQ650163

KP970754

Histone H3

JQ655115

KF563020

KF563019

KF563008

KF563007

KF563017

KP970687

KP970686

KF563026

KP970696

KP970693

KP970692

KP970691

KP970690

COI

OPU_BS_El2011-24

OPU_BS_El2011-23

OPU_BS_El2011-22

OPU_BS_El2011-21

OPU_BS_El2011-20

OPU_BS_El2010-8

OPU_BS_El2010-7

OPU_BS_El2010-4

EL2004Niugawa38

OPU_BS_Ei2011-2

OPU_BS_Ei2011-1

EI1997Kibune

OPU_BS_Ec2010-226

OPU_BS_Ea2013-58

OPU_BS_Ea2013-57

OPU_BS_Ea2013-55

OPU_BS_E2011-515

OPU_BS_E2011-514

OPU_BS_E2011-513

OPU_BS_E2013-130

OPU_BS_E2013-76

OPU_BS_E2013-74

OPU_BS_E2013-72

OPU_BS_E2013-71

OPU_BS_E2012-225

OPU_BS_E2012-132

OPU_BS_E2012-133

OPU_BS_E2012-131

OPU_BS_E2012-191

OPU_BS_E2012-190

Specimen voucher

4
K. Wakimura, et al.

imago
imago
imago
imago
imago
imago
imago
subimago
subimago
subimago
subimago
subimago
imago
imago
imago
nymph
nymph
nymph
nymph
nymph
nymph
imago
imago
imago
imago
imago
imago
imago
nymph

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus latifolium

Epeorus napaeus

Epeorus napaeus

Epeorus napaeus

Epeorus napaeus

Epeorus sp.

Epeorus sp.

Heptagenia kihada

Proepeorus nipponicus

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Stage

Epeorus latifolium

Family name Species nameNara, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Nara, Japan

Shiga, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Location

14 Dec, 201227 Apr, 2008

27 Apr, 2008

27 Apr, 2008

27 Apr, 2008

27 Apr, 2008

27 Apr, 2008

9 May, 2009

Mar, 201214 Dec, 201214 Dec, 201214 Dec, 201214 Dec, 201214 Dec, 2012

9 May, 2009

11 May, 2014

11 May, 201427 Apr, 201327 Apr, 201327 Apr, 201327 Apr, 201327 Apr, 201314 Apr, 201114 Apr, 201114 Apr, 201114 Apr, 201114 Apr, 201114 Apr, 201114 Apr, 2011

Collection Date

KF562894

GU354160

KF562875

KP970812

KF562898

KF562897

KF562896

GU354159

KP970761

KP970760

KP970759

KP970758

KF562872

JQ650178

KF562871

KF562870

18S rRNAKF562976

KP970756

KP970755

JQ650157

JQ650156

JQ650155

JQ650132

JQ650154

Histone H3

JQ319383

JQ319382

JQ319381

JQ319380

JQ319379

JQ319378

GU354164

GU354163

KP970714

KP970713

KP970700

KF563022

COI

OPU_BS_Rj2012-241

KU_Rj2008-69

KU_Rj2008-68

KU_Rj2008-67

KU_Rj2008-65

KU_Rj2008-64

Eni2009-131Nakaigawa
KU_Rj2008-63

OPU_BS_Hk2012-24

OPU_BS_E2012-236

OPU_BS_E2012-235

OPU_BS_En2012-253

OPU_BS_En2012-252

Ena2009-125Sannokou
OPU_BS_En2012-251

OPU_BS_El2014-2

OPU_BS_El2014-1

OPU_BS_El2013-143

OPU_BS_El2013-5

OPU_BS_El2013-4

OPU_BS_El2013-2

OPU_BS_El2013-1

OPU_BS_El2011-56

OPU_BS_El2011-30

OPU_BS_El2011-29

OPU_BS_El2011-28

OPU_BS_El2011-27

OPU_BS_El2011-26

OPU_BS_El2011-25

Specimen voucher

DNA barcodes of Japanese mayflies

whole bodywing muscle

sperm vesicles

wing muscle

wing muscle

sperm vesicles

wing muscle

sperm vesicles

hemolymph

whole bodywhole bodywhole bodywhole bodywhole bodysperm vesicles

leg

leg

wing muscle

sperm vesicles

wing muscle

wing muscle

wing muscle

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

wing muscle

wing muscle

Source tissue

Table 1. Accession numbers of sequence data and source information.



5

leg
leg
sperm vesicles

imago
imago
imago
imago
imago
imago
nymph
nymph
nymph
imago
imago
subimago
nymph
nymph
imago
subimago
subimago
subimago
subimago
subimago
imago
imago
imago
imago
imago
imago
imago

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena japonica

Rhithrogena minazuki

Rhithrogena minazuki

Rhithrogena minazuki

Rhithrogena tateyamana

Rhithrogena tateyamana

Rhithrogena tateyamana

Rhithrogena
tetrapunctigera
Rhithrogena
tetrapunctigera
Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

Rhithrogena sp.

sperm vesicles

wing muscle

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

wing muscle

wing muscle

wing muscle

wing muscle

wing muscle

sperm vesicles

wing muscle

wing muscle

sperm vesicles

whole bodywhole body

hemolymph

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

imago

Rhithrogena japonica

whole body

Source tissue

nymph

Stage

Rhithrogena japonica

Family name Species name

Table 1. Accession numbers of sequence data and source information.

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Saitama, Japan

Saitama, Japan

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Okinawa, Japan

Okinawa, Japan

Okinawa, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Location

5 May, 2012

5 May, 2012

5 May, 2012

5 May, 2012

5 May, 2012

5 May, 2012

5 May, 201224 Apr, 201024 Apr, 201024 Apr, 201024 Apr, 201024 Apr, 2010

25 Apr, 2009

6 Dec, 2008

6 Dec, 2008

16 Nov, 201116 Nov, 201116 Nov, 201123 Apr, 201222 Apr, 201222 Apr, 201225 Apr, 200925 Apr, 200925 Apr, 200925 Apr, 2004

24 Apr, 2005

24 Apr, 2005

24 Apr, 2005

14 Dec, 2012

Collection Date

KF562914

KF562877

KF562913

KF562912

KF562911

JQ650203

JQ650202

GU354153

KP970787

KP970786

JQ650198

KF562936

KF562935

KF562934

GU354149

GU354148

GU354147

EF680332

KF562895

18S rRNAKF562969

KF562968

KF562967

KF562966

KF562965

KF562964

KF562962

JQ650146

GQ433997

JQ650141

JQ650140

JQ650139

AY850372

KF562977

Histone H3

KF563014

KF563010

KF563013

KF563012

KP970707

KP970706

KF563058

JQ319386

JQ319385

JQ319384

COI

OPU_BS_R2012-158

OPU_BS_R2012-157

OPU_BS_R2012-156

OPU_BS_R2012-155

OPU_BS_R2012-154

OPU_BS_R2012-153

OPU_BS_R2012-102

OPU_BS_R2010-19

OPU_BS_R2010-12

OPU_BS_R2010-16

OPU_BS_R2010-15

OPU_BS_R2010-11

Rxx2009-08-Oosako-U

OPU_BS_Rt2013-359

OPU_BS_Rt2013-358

OPU_BS_Rt2011-603

OPU_BS_Rt2011-602

OPU_BS_Rt2011-601

OPU_BS_Rm2012-218

OPU_BS_Rm2012-217

OPU_BS_Rm2012-216

Rja2009-03-Oosako-U

Rja2009-02-Oosako-U

Rja2009-01-Oosako-U

RJ2004Niugawa1

OPU_Rj2005-10

OPU_Rj2005-07

OPU_Rj2005-02

OPU_BS_Rj2012-242

Specimen voucher

6
K. Wakimura, et al.

subimago
subimago
subimago
imago
nymph
nymph
nymph
imago
imago
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph

Cincticostella elongatula

Cincticostella elongatula

Cincticostella nigra

Cincticostella nigra

Cincticostella nigra

Cincticostella orientalis

Cincticostella sp.

Cincticostella sp.

Drunella basalis

Drunella basalis

Drunella basalis

Drunella basalis

Drunella basalis

Drunella basalis

Drunella basalis

Drunella basalis

Drunella basalis

Drunella basalis

Drunella cryptomeria

Drunella cryptomeria

Drunella cryptomeria

Drunella ishiyamana

Drunella kohnoi

Drunella sachalinensis

Drunella sachalinensis

Drunella sachalinensis

Drunella trispina

Drunella sp.

imago

Stage

Cincticostella elongatula

Ephemerellidae

Heptageniidae sp.

Family name Species namewhole body

Wakayama, Japan

Nara, Japan

Kanagawa, Japan

Kanagawa, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Fukuoka, Japan

Fukuoka, Japan

Fukuoka, Japan

Fukuoka, Japan

Fukuoka, Japan

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Location

15 May, 2011

14 May, 2011

Mar, 2012

Mar, 2012

15 May, 2011

5 May, 2012

15 May, 2011

5 May, 2012

5 May, 2012

5 May, 201216 Apr, 201216 Apr, 201216 Apr, 201216 Apr, 201216 Apr, 2012

Mar, 2012

Mar, 2012

Mar, 2012

Mar, 2012

Mar, 201215 Apr, 201115 Apr, 2011

14 May, 2011

14 May, 2011

15 May, 2011

8 May, 201015 Apr, 201115 Apr, 201115 Apr, 201128 Apr, 2013

Collection Date

KP970806

JQ650193

KF562903

KF562902

KF562868

KF562937

KF562866

KP970809

KP970808

KP970807

KF562886

KF562885

JQ650189

JQ650195

JQ650194

JQ650218

JQ650210

JQ650191

JQ650186

JQ650185

KP970781

18S rRNA

KP970750

KF562990

KF562989

KF563000

JQ650124

KP970753

KP970752

KP970751

KF562974

KF563005

JQ650161

JQ650127

JQ650126

JQ650123

JQ650121

JQ650136

Histone H3

KF563041

KF563040

KF563046

KF563045

KF563044

KF563043

KF563042

KF563037

KF563036

KF563035

KF563034

KF563033

COI

OPU_BS_D2011-166

OPU_BS_Dt2011-255

OPU_BS_Ds2012-55

OPU_BS_Ds2012-54

OPU_BS_Di2011-170

OPU_BS_Dk2012-220

OPU_BS_Di2011-165

OPU_BS_D2012-224

OPU_BS_D2012-223

OPU_BS_D2012-222

OPU_BS_Db2012-164

OPU_BS_Db2012-161

OPU_BS_Db2012-160

OPU_BS_Db2012-101

OPU_BS_Db2012-100

OPU_BS_Db2012-7

OPU_BS_Db2012-6

OPU_BS_Db2012-5

OPU_BS_Db2012-2

OPU_BS_Db2012-1

OPU_BS_C2011-117

OPU_BS_C2011-113

OPU_BS_Co2011-309

OPU_BS_Cn2011-308

OPU_BS_ Cn2011-171

OPU_BS_Cn2010-363

OPU_BS_Ce2011-128

OPU_BS_Ce2011-99

OPU_BS_Ce2011-98

OPU_BS_H2013-235

Specimen voucher

DNA barcodes of Japanese mayflies

hemolymph

whole body

hemolymph

hemolymph

wing muscle

whole bodywing muscle

wing muscle

wing muscle

wing muscle

hemolymph

hemolymph

hemolymph

hemolymph

hemolymph

hemolymph

hemolymph

hemolymph

hemolymph

wing muscle

wing muscle

hemolymph

hemolymph

hemolymph

wing muscle

sperm vesicles

sperm vesicles

wing muscle

wing muscle

Source tissue

Table 1. Accession numbers of sequence data and source information.



7

hemolymph

nymph
nymph
nymph
nymph
nymph

Drunella sp.

Drunella sp.

Drunella sp.

Drunella sp.

Drunella sp.
hemolymph

nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
imago
nymph
nymph
nymph
imago
imago
nymph
nymph
nymph
nymph
nymph
nymph
nymph

Ephemerella atagosana

Ephemerella atagosana

Ephemerella atagosana

Ephemerella atagosana

Ephemerella atagosana

Ephemerella notata

Ephemerella notata

Ephemerella notata

Ephemerella notata

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Ephemerella sp.

Serratella setigera

whole body

abdomenabdomenabdomenabdomenabdomenabdomensperm vesicles

wing muscle

hemolymph

hemolymph

hemolymph

sperm vesicles

leg

leg

leg

leg

whole body

hemolymph

hemolymph

whole body

hemolymph

Ephacerella longicaudata nymph

hemolymph

hemolymph

hemolymph

hemolymph

hemolymph

nymph

Drunella sp.

wing muscle

Source tissue

nymph

Stage

Drunella sp.

Family name Species name

Table 1. Accession numbers of sequence data and source information.

Nara, Japan

Osaka, Japan

Osaka, Japan

Osaka, Japan

Osaka, Japan

Osaka, Japan

Osaka, Japan

Nara, Japan

Wakayama, Japan

Kyushu, Japan

Kyushu, Japan

Kyushu, Japan

Nara, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Shiga, Japan

Shiga, Japan

Shiga, Japan

Fukuoka, Japan

Fukuoka, Japan

Fukuoka, Japan

Fukuoka, Japan

Fukuoka, Japan

Kanagawa, Japan

Kanagawa, Japan

Location

15 May, 201129 Jun, 201429 Jun, 201429 Jun, 201429 Jun, 201429 Jun, 201429 Jun, 201427 Apr, 2013

5 May, 2012

Mar, 2012

Mar, 2012

Mar, 201225 Apr, 200913 Apr, 201413 Apr, 2014

11 Apr, 2014

11 Apr, 2014

Mar, 2012

Mar, 2012

Mar, 2012

Mar, 2012

Mar, 2012

Mar, 201215 Apr, 201215 Apr, 201215 Apr, 201215 Apr, 201215 Apr, 2012

Mar, 2012

Mar, 2012

Collection Date

KP970804

KP970803

KP970802

KP970801

KP970800

KF562905

KF562904

GU354155

KP970799

KP970798

KF562893

KF562892

KF562889

KF562888

KF562887

KF562921

KF562920

KF562919

KF562918

KF562917

KF562907

KF562906

18S rRNAJQ650125

KP970729

KF562963

KF562992

KF562959

KF562991

KF562988

KF562987

KF562986

KF562984

KF562983

KF562982

KF562961

KF562960

Histone H3

KP970728

KP970727

KP970726

KP970725

KF563051

KF563054

KF563053

KP970724

KP970723

KP970722

KF563052

KF563039

KF563038

KF563050

KF563049

KF563048

KF563055

COI

OPU_BS_Ss2011-167

OPU_BS_E2014-76

OPU_BS_E2014-73

OPU_BS_E2014-72

OPU_BS_E2014-71

OPU_BS_E2014-69

OPU_BS_E2014-68

OPU_BS_E2013-51

OPU_BS_E2012-129

OPU_BS_E2012-91

OPU_BS_E2012-90

OPU_BS_E2012-86

Exx2009-16-Ootaki-D

OPU_BS_En2014-23

OPU_BS_En2014-22

OPU_BS_En2014-18

OPU_BS_En2014-17

OPU_BS_Ea2012-45

OPU_BS_Ea2012-44

OPU_BS_Ea2012-43

OPU_BS_Ea2012-34

OPU_BS_Ea2012-33

OPU_BS_El2012-18

OPU_BS_D2012-182

OPU_BS_D2012-181

OPU_BS_D2012-180

OPU_BS_D2012-179

OPU_BS_D2012-178

OPU_BS_D2012-97

OPU_BS_D2012-96

Specimen voucher

8
K. Wakimura, et al.

Baetidae

subimago
subimago
subimago
subimago
subimago
subimago
nymph
nymph
nymph
imago
imago

Alainites yoshinensis

Baetiella japonica

Baetiella japonica

Baetiella japonica

Baetiella japonica

Baetis thermicus

Baetis thermicus

Baetis thermicus

Baetis sp.

Baetis sp.

subimago

Ephemerellidae sp.

Alainites yoshinensis

subimago

Ephemerellidae sp.

nymph

subimago

Ephemerellidae sp.

subimago

subimago

Ephemerellidae sp.

Alainites yoshinensis

nymph

Uracanthella punctisetae

Acentrella sibirica

nymph

Uracanthella chinoi

nymph

nymph

Uracanthella chinoi

Acentrella sibirica

nymphTorleya japonica

nymph

nymph

Torleya japonica

Acentrella gnom

nymph

Teloganopsis brocha

nymph

nymph

Acentrella gnom

nymph

Teloganopsis brocha

Stage

Serratella setigera

Family name Species namewing muscle

sperm vesicles

whole bodywhole bodywhole bodywing muscle

sperm vesicles

wing muscle

sperm vesicles

wing muscle

wing muscle

whole bodythoraxwhole body

abdomenthoraxleg

wing muscle

wing muscle

wing muscle

sperm vesicles

leg

leg

hemolymph

hemolymph

whole bodywhole bodywhole body

Source tissue

Table 1. Accession numbers of sequence data and source information.

Wakayama, Japan

Nara, Japan

Nara, Japan

Kanagawa, Japan

Kanagawa, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

Kanagawa, Japan

Nara, Japan

Nara, Japan

Kyoto, Japan

Kyoto, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Okayama, Japan

Okayama, Japan

Okinawa, Japan

Okinawa, Japan

TaiwanTaiwanWakayama, Japan

Location

24 Apr, 2010

25 Apr, 200914 Dec, 201216 Nov, 2011

9 Nov, 201124 Apr, 201024 Apr, 201024 Apr, 201024 Apr, 201027 Apr, 201327 Apr, 2013

9 Nov, 201127 Apr, 201314 Dec, 2012

20 Sep, 2010

20 Sep, 2010

11 May, 201427 Apr, 201327 Apr, 201327 Apr, 2013

25 Apr, 200916 Sep, 201216 Sep, 201222 Apr, 201222 Apr, 2012

Sep, 2011

Sep, 201116 Nov, 2011

Collection Date

GU354152

KF562900

JQ650200

JQ650211

KF562874

KF562899

KP970789

KP970788

KP970779

KP970778

KP970777

GU354154

KP970785

KP970784

KF562926

KF562925

KF562873

JQ650199

18S rRNAJQ655111

KF562973

KF562972

JQ650151

JQ650150

JQ650149

KF562980

KP970739

GQ433998

KP970738

KF562995

KF562957

KF562956

JQ650142

Histone H3

KF563031

KF563030

KF563016

KF563015

KP970698

KP970697

KP970701

KP970710

KP970709

KP970715

KP970705

KP970704

JQ655119

JQ655113

COI

OPU_BS_B2010-23

Bxx2009-07-Oosako-U

OPU_BS_Bt2012-261

OPU_BS_Bt2011-606

OPU_BS_Bt2011-564

OPU_BS_Bj2010-41

OPU_BS_Bj2010-40

OPU_BS_Bj2010-39

OPU_BS_Bj2010-38

OPU_BS_Ay2013-132

OPU_BS_Ay2013-131

OPU_BS_Ay2011-566

OPU_BS_As2013-145

OPU_BS_As2012-254

OPU_BS_Ag2013-362

OPU_BS_Ag2013-361

OPU_BS_E2014-4

OPU_BS_E2013-169

OPU_BS_E2013-168

OPU_BS_E2013-167

Uru2009-10-Oosako-D

OPU_BS_Uc2013-357

OPU_BS_Uc2013-356

OPU_BS_Tj2012-194

OPU_BS_Tj2012-193

OPU_BS_Tb2011-543

OPU_BS_Tb2011-542

OPU_BS_Es2011-605

Specimen voucherDNA barcodes of Japanese mayflies

9Ephemeridae

nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
imago
imago

Labiobaetis atrebatinus
orientalis
Labiobaetis tricolor

Labiobaetis tricolor

Nigrobaetis chocoratus

Nigrobaetis sp. D

Nigrobaetis taiwanensis

Nigrobaetis taiwanensis

Tenuibaetis flexifemora

Tenuibaetis flexifemora

Baetidae sp.

Baetidae sp.

nymph

imago

Baetis sp.

Ephemera japonica

subimago

Baetis sp.

nymph

subimago

Baetis sp.

Ephemera japonica

imago

Baetis sp.

nymph

subimago

Baetis sp.

Ephemera japonica

subimago

Baetis sp.

imago

imago

Baetis sp.

imago

imago

Baetis sp.

Ephemera formosana

thoraximago

Baetis sp.

Ephemera formosana

whole body

imago

Baetis sp.

whole bodywhole bodywhole bodysperm vesicles

wing muscle

leg

leg

thoraxthoraxwhole bodywhole bodywhole bodywhole bodythoraxthoraxwing muscle

sperm vesicles

wing muscle

wing muscle

thoraxwhole bodywing muscle

sperm vesicles

wing muscle

wing muscle

imago

Baetis sp.

wing muscle

Source tissue

imago

Stage

Baetis sp.

Family name Species name

Table 1. Accession numbers of sequence data and source information.

Miyagi, Japan

Miyagi, Japan

Miyagi, Japan

TaiwanTaiwanWakayama, Japan

Wakayama, Japan

Kyoto, Japan

Kyoto, Japan

Kanagawa, Japan

Kanagawa, Japan

Wakayama, Japan

Wakayama, Japan

Chiba, Japan

Chiba, Japan

Kanagawa, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Location

8 Aug, 2010

8 Aug, 2010

8 Aug, 2010

Sep, 2011

Sep, 2011

11 May, 2014

11 May, 2014

20 Sep, 2010

20 Sep, 2010

25 Nov, 2011

25 Nov, 2011

5 May, 2012

5 May, 2012

9 May, 2009

9 May, 200924Nov, 201127 Apr, 201327 Apr, 201327 Apr, 201327 Apr, 201327 Apr, 201327 Apr, 201314 May, 201115 Apr, 201114 Apr, 201114 Apr, 201124 Apr, 201024 Apr, 2010

Collection Date

JQ650224

JQ650223

KP970791

KP970790

KF562883

KF562884

KF562939

KF562940

KP970782

KF562878

KP970776

KP970771

KP970770

JQ650192

JQ650182

JQ650173

JQ650208

18S rRNAJQ650169

JQ650168

JQ650167

JQ650164

KP970741

KP970740

KF563002

KF562975

KP970735

KP970734

KF562950

JQ650138

JQ650158

JQ650129

JQ655112

Histone H3

JQ655117

KP970717

KP970716

KP970712

KP970711

KF563059

KF563060

KF563032

KP970699

KP970695

KP970694

JQ655116

COI

OPU_BS_Ej2010-EJ3

OPU_BS_Ej2010-EJ2

OPU_BS_Ej2010-EJ1

OPU_BS_Ef2011-526

OPU_BS_Ef2011-525

OPU_BS_B2014-6

OPU_BS_B2014-5

OPU_BS_Tf2013-364

OPU_BS_Tf2013-363

OPU_BS_Bt2011-608

OPU_BS_Bt2011-607

OPU_BS_N2012-226

OPU_BS_Nc2012-227

OPU_BS_Lt2013-353

OPU_BS_Lt2013-352

OPU_BS_La2011-614

OPU_BS_B2013-166

OPU_BS_B2013-134

OPU_BS_B2013-103

OPU_BS_B2013-102

OPU_BS_B2013-101

OPU_BS_B2013-85

OPU_BS_B2011-494

OPU_BS_B2011-147

OPU_BS_B2011-74

OPU_BS_B2011-19

OPU_BS_B2010-23
clone 2
OPU_BS_B2010-24

Specimen voucher

10
K. Wakimura, et al.

nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
imago
imago
imago
imago
nymph
nymph

Ephemera japonica

Ephemera japonica

Ephemera japonica

Ephemera japonica

Ephemera japonica

Ephemera japonica

Ephemera orientalis

Ephemera orientalis

Ephemera strigata

Ephemera strigata

Ephemera strigata

Ephemera strigata

Ephemera strigata

Ephemera strigata

sperm vesicles
sperm vesicles
wing muscle
sperm vesicles
sperm vesicles
sperm vesicles
sperm vesicles
whole bodywhole bodywhole body

Paraleptophlebia chocolataimago

Paraleptophlebia japonica subimago

Paraleptophlebia japonica subimago

Paraleptophlebia spinosa imago

Paraleptophlebia spinosa imago

Paraleptophlebia spinosa imago

Paraleptophlebia spinosa imago

Paraleptophlebia westoni nymph

Paraleptophlebia westoni nymph

Paraleptophlebia westoni nymph

whole bodysperm vesicles

nymph

Choroterpides nigellawhole bodywhole bodywhole bodysperm vesicles

sperm vesicles

sperm vesicles

sperm vesicles

wing muscle

wing muscle

wing muscle

wing muscle

wing muscle

wing muscle

wing muscle

wing muscle

hemolymph

whole body

Source tissue

Paraleptophlebia chocolataimago

nymph

Choroterpides nigella

Leptophlebiidae

nymphEphemera japonica

Stage

Ephemera japonica

Family name Species name

Table 1. Accession numbers of sequence data and source information.

12 Sep, 201112 Sep, 201112 Sep, 201114 Apr, 201114 Apr, 2011

9 Apr, 2006

9 Apr, 2006

24 Apr, 201024 Apr, 2010

25 Apr, 200925 Apr, 2009

Sep, 2011

Sep, 2011

8 Aug, 2010

8 Aug, 2010

25 Apr, 2009

Mar, 2012

Mar, 201211 Sep, 2011

11 Sep, 2011

11 Sep, 2011

11 Sep, 2011

11 Sep, 2011

11 Sep, 201112 Sep, 201112 Sep, 2011

Collection Date

JQ650219

JQ650181

JQ650180

GU354146

JQ650205

JQ650204

JQ650225

GU354151

EF680329

EF680328

KF562891

KF562890

KF562948

KF562947

KF562946

KF562945

KF562944

KF562943

JQ650197

18S rRNAKF562952

KF562951

JQ650135

JQ650134

EF055451

JQ650145

JQ650144

GQ434000

GQ433999

JQ650166

JQ650165

JQ650171

JQ650170

AY860841

KF562958

KF562985

JQ650153

Histone H3

KF563011

GU354161

JQ655118

JQ655114

COI

OPU_BS_Pw2011-502

OPU_BS_Pw2011-496

OPU_BS_Pw2011-495

OPU_BS_Ps2011-42

OPU_BS_Ps2011-41

PS2006Niugawa 1-2-3

PS2006Niugawa-1

OPU_BS_Pj2010-14

OPU_BS_Pj2010-13

Pch2009-18-Ootaki-D

Pch2009-17-Ootaki-D

OPU_BS_Cn2011-539

OPU_BS_Cn2011-534

OPU_BS_Es2010-ES3

OPU_BS_Es2010-ES2

Est2009-06-Oosako-U

ES2002Kyoto

ES2002Kyoto2

ES2002Kyoto1

OPU_BS_Eo2012-36

OPU_BS_Eo2012-35

OPU_BS_Ej2012-270

OPU_BS_Ej2012-269

OPU_BS_Ej2012-268

OPU_BS_Ej2012-267

OPU_BS_Ej2012-266

OPU_BS_Ej2012-265

OPU_BS_Ej2011-508

OPU_BS_Ej2011-507

Specimen voucher

DNA barcodes of Japanese mayflies

Kanagawa, Japan

Kanagawa, Japan

Kanagawa, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

TaiwanTaiwanMiyagi, Japan

Miyagi, Japan

Nara, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Shiga, Japan

Shiga, Japan

Fukushima, Japan

Fukushima, Japan

Fukushima, Japan

Fukushima, Japan

Fukushima, Japan

Fukushima, Japan

Kanagawa, Japan

Kanagawa, Japan

Location

11imagosubimago
subimago
imago
imago
imago
subimago
nymph
nymph

Ameletus montanus

Ameletus montanus

Ameletus sp.

Ameletus sp.

Ameletus sp.

Ameletus sp.

Ameletus sp.

Ameletus sp.

nymph

Thraulus grandis

Ameletus montanus

nymphThraulus grandis

imago

nymph

Thraulus grandis

Ameletus montanus

nymphThraulus grandis

imago

subimago

Paraleptophlebia sp.

Ameletus costalis

imago

Paraleptophlebia sp.

imago

subimago

Paraleptophlebia sp.

Ameletus costalis

imago

Paraleptophlebia sp.

imago

nymph

Paraleptophlebia sp.

Ameletus costalis

nymphParaleptophlebia sp.

nymph
nymph

Potamanthus formosus

Potamanthus formosus

Potamanthidae

Ameletidae

wing muscle

leghemolymph

legwhole bodywing muscle

wing muscle

wing muscle

sperm vesicles

wing muscle

wing muscle

ovary

sperm vesicles

wing muscle

wing muscle

sperm vesicles

whole body

hemolymph

hemolymph

hemolymph

sperm vesicles

sperm vesicles

wing muscle

wing muscle

whole bodywhole bodywing muscle

subimago

Paraleptophlebia sp.

Source tissue

Stage

Paraleptophlebia westoni imago

Family name Species name

Table 1. Accession numbers of sequence data and source information.

Kyoto, Japan

Fukuoka, Japan

Kyoto, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Kyoto, Japan

Okinawa, Japan

Okinawa, Japan

Okinawa, Japan

Okinawa, Japan

Wakayama, Japan

Wakayama, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Nara, Japan

Kanagawa, Japan

Location

11 Apr, 201415 Apr, 2012

11 Apr, 201414 Dec, 201215 Apr, 201015 Apr, 201124 Apr, 201024 Apr, 201027 Apr, 201327 Apr, 2013

25 Apr, 200925 Apr, 200915 Apr, 201114 Apr, 2011

Apr, 1997

23 Apr, 2012

23 Apr, 2012

23 Apr, 2012

23 Apr, 201228 Apr, 201328 Apr, 201327 Apr, 201327 Apr, 201314 Dec, 201214 Dec, 2012

8 May, 2010

12 Sep, 2011

Collection Date

KP970795

KF562924

KF562901

JQ650190

JQ650187

JQ650201

JQ650215

KP970766

KP970765

GU354150

JQ650188

JQ650179

EF680323

KF562933

KF562932

KF562931

KF562930

KP970780

KP970773

KP970772

JQ650209

JQ650196

18S rRNA

KP970748

KF562994

KP970747

KF562981

JQ650122

JQ650137

JQ650143

JQ650152

KP970730

GQ433996

GQ433995

JQ650160

JQ650133

AY870291

KF562999

KF562998

KF562997

KF562996

KP970733

KP970732

KP970731

KF562979

KF562978

JQ650148

Histone H3

KF563056

KF563009

KP970689

KP970688

KF563021

KF563057

KF563018

KF563025

COI

OPU_BS_Pf2014-13

OPU_BS_Pf2012-192

OPU_BS_A2014-12

OPU_BS_A2012-262

OPU_BS_A2011-122

OPU_BS_A2011-106

OPU_BS_A2010-10

OPU_BS_A2010-9

OPU_BS_Am2013-68

Amo2009-04Oosako-U
Amo2009-05Oosako-U
OPU_BS_Am2013-67

OPU_BS_Ac2011-108

OPU_BS_Ac2011-38

AC1997Kibune-O

OPU_BS_Tg2012-214

OPU_BS_Tg2012-212

OPU_BS_Tg2012-211

OPU_BS_Tg2012-210

OPU_BS_P2013-202

OPU_BS_P2013-200

OPU_BS_P2013-106

OPU_BS_P2013-105

OPU_BS_P2012-249

OPU_BS_P2012-248

OPU_BS_P2010-362

OPU_BS_Pw2011-504

Specimen voucher

12
K. Wakimura, et al.

nymph
nymph
nymph
nymph
nymph
nymph

Siphlonurus sanukensis

Siphlonurus sp.

Siphlonurus sp.

Siphlonurus sp.

Siphlonurus sp.

Siphlonurus sp.

Isonychia japonica
nymph
nymph

Ephoron shigae

Ephoron shigae

nymph

imago

nymph

Caenis sp.

nymph

Siphlonurus sanukensis

imago

Potamanthus idiocerus

Siphlonurus sanukensis

imago

Polymitarcyidae

Isonychiidae

CaenidaeSiphlonuridae

imago

Potamanthus idiocerus

Stage

Potamanthus formosus

Family name Species namethoraxthoraxwing muscle

whole body

leg

leg

leg

leg

leg

hemolymph

hemolymph

wing muscle

wing muscle

sperm vesicles

leg

Source tissue

Table 1. Accession numbers of sequence data and source information.

Okayama, Japan

Okayama, Japan

Wakayama, Japan

Nara, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Kyoto, Japan

Fukuoka, Japan

Fukuoka, Japan

Fukuoka, Japan

TaiwanTaiwanNara, Japan

Location

16 Sep, 201116 Sep, 2011

15 May, 2011

14 May, 201111 Apr, 2014

11 Apr, 2014

11 Apr, 2014

11 Apr, 2014

11 Apr, 201416 Apr, 201216 Apr, 201216 Apr, 2012

Sep, 2011

Sep, 2011

6 Jul, 2014

Collection Date

KP970783

KF562949

KP970805

KP970794

KP970793

KP970792

KF562916

KF562915

JQ650222

18S rRNA

KP970737

KP970736

KF563006

KP970757

KP970746

KP970745

KP970744

KP970743

KP970742

KF562971

KF562993

KF562970

KF562955

KF562954

KP970749

Histone H3

KP970703

KP970702

KP970721

KP970720

KP970719

KP970718

KF563047

KF563029

COI

OPU_BS_Es2013-355

OPU_BS_Es2013-354

OPU_BS_Ij2011-169

OPU_BS_C2011-493

OPU_BS_S2014-11

OPU_BS_S2014-10

OPU_BS_S2014-9

OPU_BS_S2014-8

OPU_BS_S2014-7

OPU_BS_Ss2012-176

OPU_BS_Ss2012-175

OPU_BS_Ss2012-174

OPU_BS_Pi2011-522

OPU_BS_Pi2011-521

OPU_BS_Pf2014-77

Specimen voucherDNA barcodes of Japanese mayflies

13

14

K. Wakimura, et al.

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

Sequence

TargetAnnealing 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

3 Results and discussion
A 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, fromwhich 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 notdetermined. 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 madeto analyze the unsequenced species). A portion of the DNA
sequences (98 among 486 sequences) were not registeredwith full scientific names due to our conservative way ofidentification, 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 futurestudies, 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 resultof long branch attraction [14] due to the highly variable
nature of these genes, or saturation of substitutions at thethird 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 clusteringof families (Fig. 1C) when the highly variable V4 stem-loop
regions (containing many insertions/deletions that were
impossible to align unambiguously) were excluded fromthe analysis.

The specimen of Nigrobaetis sp. D (N2012-226) wasrecovered 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 H3tree (Fig. 1B) it is placed well within a clade comprising
Empherellidae specimens, as sister-group to Ephemerallasp. (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, howeveras 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 ininferring phylogenetic relationship among families but
were useful in identifying species (i.e., DNA barcoding).
In contrast, the more conserved 18S rRNA gene was usefulin confirming the integrity of the source DNA samples(e.g. contamination) and was adequately informativefor elucidating inter-family divergence, but not suitable
for species identification. We also found intraspecificvariations in COI sequences of some species (Fig. 2), thatcan be applicable to characterizing the genetic structuresof 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, whichwere morphologically identified. Our results indicated that
COI, histone H3 gene sequences could serve as markers
for species identification, whereas more conserved 18SrRNA 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 bycomparing the haplotype frequency of certain species in
the future.



DNA barcodes of Japanese mayflies

15

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 extraction (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.

16

K. Wakimura, et al.

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 extraction (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.

DNA barcodes of Japanese mayflies

17

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 extraction (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.

18

K. Wakimura, et al.

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

help and support in collecting mayfly samples in KiiNyugawa 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).

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 and2012-55; 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.



DNA barcodes of Japanese mayflies

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 and2012-55; 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.

19

20

K. Wakimura, et al.

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 and2012-55; 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.



DNA barcodes of Japanese mayflies

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 and2012-55; 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.

21

22

K. Wakimura, et al.

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 and2012-55; 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.



DNA barcodes of Japanese mayflies

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 and2012-55; 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.

23

24

K. Wakimura, et al.

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 and2012-55; 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.



Conflict of interest: Authors declare nothing to disclose.

References
[1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

Resh, V.H., Freshwater benthic macroinvertebrates andrapid assessment procedures for water quality monitoring indeveloping and newly industrialized countries. pp.167-177. In:
Biological Assessment and Criteria - Tools for Water Resource
Planning and Decision Making (Davis, W.S. and Simon, T.P.
editors). 1995, Lewis Publishers, London, ISBN 0-87371-894-1,
420 pp.
Baunernfeind, E. & Moog, O., Mayflies (Insecta:
Ephemeroptera) and the assessment of ecological integrity:
a methodological approach, Hydrobiologia, 2000, 422 /423,
71-83. DOI: 10.1023/A:1017090504518
Edsall, T. A., Bur, M. T., Gorman, O. T. & Schaeffer, J. S.,
Burrowing mayflies as indicators of ecosystem health: Status of
populations in western Lake Erie, Saginaw Bay and Green Bay,
Aquatic Ecosystem Health & Management, 2005, 8, 107-116.
DOI: 10.1080/14634980590953194
Iwasaki, Y., Kagaya, T., Miyamoto, K.-I. & Matsuda, H., Effectsof heavy metals on riverine benthic macroinvertebrate
assemblages with reference to potential food availability for
drift-feeding fishes, Environmental Toxicology and Chemistry,
2009, 28, 354-363 DOI: 10.1897/08-200.1
Tautz, D., Arctander, P., Minelli, A., Thomas, R. H. & Vogler, A.
P., A plea for DNA taxonomy, Trends in Ecology and Evolution,
2003, 18, 70-74. DOI: 10.1016/S0169-5347(02)00041-1
Hebert, P. D. N., Cywinska, A., Ball, S. L., & de Waard, J. R.,
Biological identifications through DNA barcodes, Proceedingsof Royal Society of London B, 2003, 270, 313-321. DOI:
10.1098/rspb.2002.2218
Ishiwata, S.-I. & Takemon, Y., Checklist of Japanese names ofJapanese mayflies with notes on nomenclature (in Japanese),Japanese Journal of Limnology, 2005, 66, 11-35. DOI: 10.3739/
rikusui.66.11
Ogden, T. H. & Whiting, M. F., Phylogeny of Ephemeroptera(mayfies) based on molecular evidence, MolecularPhylogenetics and Evolution, 2005, 37, 625-643. DOI:10.1016/j.
ympev.2005.08.008
Misof, B., Niehuis, O., Bischoff, I., Rickert, A., Erpenbeck,
D. & Staniczek, A., A hexapod nuclear SSU rRNA secondarystructure model and catalog of taxon-specific structuralvariation, Journal of Experimental Zoology (Molecular andDNA barcodes of Japanese mayflies

25

Developmental Evolution) 2006, 306B, 70-88. DOI: 10.1002/
jez.b[10] Takemon, Y., Yamamoto, A., Nakashima, M., Tanida, K., Kishi,
M. & Kato, M., Isolation of sperm vesicles from adult malemayflies and other insects to prepare high molecular weight
genomic DNA samples, Molecular Biology Report, 2006, 33,
65-70. DOI: 10.1007/s11033-006-6258-4[11] Hall, T. A., BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows 95/98/NT,
Nucleic Acids Symposium Series 1999, 41, 95-98.
[12] Saitou, N. & Nei, M., The neighbor-joining method: a new
method for reconstructing phylogenetic trees, Molecular
Biology and Evolution, 1987, 4, 406-425.
[13] Felsenstein, J., PHYLIP (Phylogeny Inference Package) version
3.5c, Distributed by the author, 1993, Department of Genetics,
Univ. of Washington, Seattle.
[14] Felsenstein, J. Cases in which parsimony or compatibility
methods will be positively misleading, Systematic Zoology,
1978, 27, 401-410.
[15] Luo, A., Zhang, A., Ho, S. Y. W., Xu, W., Zhang, Y., Shi, W.,
Cameron, S. L. & Zhu, C., Potential efficacy of mitochondrial
genes for animal DNA barcoding: a case study using eutherianmammals, BMC Genomics, 2011, 12, 84. DOI: 10.1186/14712164-12-84[16] Ball, S. L. & Hebert, P. D. N., Biological identifications ofmayflies (Ephemeroptera) using DNA barcodes, Journalof North American Society, 2005, 24 (3), 508-524. DOI:
10.1899/04-142.1[17] Cardoni, S., Tenchini, R., Ficulle, I., Piredda, R., Simeone, M.,
C. & Belfiore, C., DNA barcode assessment of Mediterranean
mayflies (Ephemeroptera), benchmark data for a regionalreference library for rapid biomonitoring of freshwaters,
Bochemical Systematics and Ecology, 2015, 62, 36-50. DOI:
10.1016/j.bse.2015.07.035[18] Ogden, T. H. & Whiting, M. F., The problem with “the Paleoptera
Problem:” sense and sensitivity, Cladistics, 2003, 19, 432-442.
DOI: 10.1016/S0748-3007(03)00083-5[19] Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R.,
DNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebrates,
Molecular Marine Biology and Biotechnology, 1994, 3,
294–299.
Supplemental Material: The online version of this article(DOI: 10.1515/dna-2016-0001) offers supplementary material.