ZooKeys 1206: 181-190 (2024)

= / ooKeys DOI: 10.3897/zookeys.1206.124932
Research Article

DNA barcoding reveals a taxonomic fraud: Note on validity of
Propomacrus muramotoae (Coleoptera, Scarabaeidae)

Seunghyun Lee’?®, Seulmaro Hwang3, Minhyeuk Lee**®, Jinbae Seung®®, Woong Choi®, Ming Bai'’”®

Key Laboratory of Animal Biodiversity Conservation and Integrated Pest Management, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
Department of Life Sciences, Natural History Museum, London, UK

Department of Science Contents, Visang Education, Gwacheon, Republic of Korea

National Institute of Agricultural Sciences, Wanju, Republic of Korea

Insect Biosystematics Laboratory, Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea

305-403, Sechangnamsunhwan-ro, Namdong-gu, Incheon, Republic of Korea

University of Chinese Academy of Sciences, Beijing, China

Corresponding author: Ming Bai (baim@ioz.ac.cn)

N DOD oo FBP WO NY —

Abstract

Until the early 2000s, the genus Propomacrus was known to comprise two species,
occurring in the Eastern Mediterranean and Southeast China. The discovery of Pro-
pomacrus muramotoae Fujioka in Tibet and subsequently in Bhutan and Nepal, might
play a crucial role in bridging the geographical distribution gap of the Euchirini tribe
between the Mediterranean and Central China, offering profound insights into its evo-
lution and biogeography. However, all specimens, including the holotype specimen,
were sourced from a single insect vendor, with no further specimens found or cata-
logued in museum collections thereafter. During our examination of a P muramotoae
oven (acess specimen from a private collection in South Korea, we found its COI gene sequence
to be identical to that of P bimucronatus (Pallas) from Turkey, a species known for
its wide distribution and genetic variability across regional populations. This overlap

Academic editor: Andrey Frolov in genetic identity raised significant doubts, further compounded by our detection
Received: 8 April 2024 of deliberate modifications in essential diagnostic features during morphological ex-
Accepted: 28 May 2024 amination. All three specimens we examined showed crude modifications, including
Published: 8 July 2024 staining and artificial grinding. Despite our inability to access the P muramotoae type

specimens for direct examination—a challenge we attempted to overcome through

ZooBank: https://zoobank. ee eee
pS various means—it is evident that significant fraudulent tampering has occurred with

org/60626DA3-9459-42E0-ACES-

7931 F05COD8B the P muramotoae specimens. Therefore, a new synonymy is proposed: Propomacrus

bimucronatus Pallas, 1781 = P muramotoae Fujioka, 2007 (syn. nov.). We also advo-
Citation: Lee S, Hwang S, Lee M, cate for a straightforward verification of the type specimen through molecular analy-
Seung J, Choi W, Bai M (2024) DNA sis of the COI barcode region and morphological re-examination under a microscope
barcoding reveals a taxonomic fraud: for those who have access to the type specimens.

Note on validity of Propomacrus
muramotoae (Coleoptera,
Scarabaeidae). ZooKeys 1206:
181-190. https://doi.org/10.3897/
zookeys.1206.124932

Key words: DNA barcoding, Euchirini, long-armed scarab, manipulated specimen, new
synonymy

Copyright: © Seunghyun Lee et al.

This is an open access article distributed under
terms of the Creative Commons Attribution
License (Attribution 4.0 International - CC BY 4.0).

181

Seunghyun Lee et al.: Validity of Propomacrus muramotoae

Introduction

The beetle tribe Euchirini is characterized by their large size and notably elongat-
ed forelegs in males (Young 1989). These species are distributed widely across
the Mediterranean, Indo-Asian Continental, and Southwest Pacific Insular regions
(Young 1989). The tribe encompasses three genera: Cheirotonus Hope, 1841;
Euchirus Burmeister & Schaum, 1840; and Propomacrus Newman, 1837. While
Euchirus are confined to Southeast Asia and Cheirotonus spans Southeast Asia
to the Indo-Himalayan region, Propomacrus exhibits the broadest yet distinctly
disjunct distribution (Young 1989). Propomacrus bimucronatus (Pallas 1781),
originally described from Turkey, has been recorded across a range of countries
including Macedonia, Bulgaria, Greece, Turkey, Syria, Lebanon, Israel, Iran and Iraq
(Young 1989; Muramoto 2012; Bezdék 2016; Ibrahim and Fayg 2022). Propoma-
crus davidii Deyrolle, 1874, was described from central China, with its distribution
is still restricted to that region. Alexis and Makris (2002) described Propomacrus
cypriacus, distinguishing it by male protibiae shape and ornamentation, which
was later relegated to a subspecies based on mitochondrial DNA analyses, mor-
phological reassessment and ecological data (Sfenthourakis et al. 2017).

A notable discovery within this genus was Propomacrus muramotoae Fujioka,
2007, found in Tibet, with subsequent findings from Bhutan and Nepal (Muramoto
2012). This species would bridge the distribution gap between the Mediterranean
and Central China, offering invaluable insights into the evolution and biogeography
of Propomacrus and the tribe Euchirini. However, P muramotoae was morpholog-
ically similar to P bimucronatus and was described based on subtle morpholog-
ical characters like the blunt lateral margin of the pronotum and a ventral groove
on the abdomen (Fujioka 2007). Also, all specimens of P muramotoae, including
the type specimens, were obtained from Li Jingke, a beetle collector and seller
known for altering locality labels (see Discussion). These circumstances raised
significant doubts about the validity of this species, particularly given the absence
of any subsequent findings. On the other hand, Sfenthourakis et al. (2017) pro-
vided extensive genetic resources for P bimucronatus and a few DNA sequences
of other Euchirini species, revealing broad genetic variation within P bimucrona-
tus and even the P. b. cypriacus population from Cyprus displayed multiple CO!
haplotypes. This suggests that confirming the validity of P muramotoae could be
straightforward with sequencing and comparison to existing public sequences.

This study aims to clarify the status of P muramotoae by analyzing samples
labeled as being collected from “Tibet” and “Nepal”.

Material and methods

Three P muramotoae specimens, deposited in the second author’s (S.H.) col-
lection, one pair labeled as being collected from “Tibet” and one from “Nepal”,
were used in this study. Specimens were examined with an Olympus SZ61 ste-
reomicroscope and photographed with a DMC 5400 digital camera attached to
a Leica Z16 APO motorized macroscope. Serial images were combined using
Zerene Stacker.

Genomic DNA from all three samples was extracted from both thorac-
ic muscle and labial palpi of each specimen, using the DNeasy Blood & Tis-
sue kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. The

ZooKeys 1206: 181-190 (2024), DOI: 10.3897/zookeys.1206.124932 182

Seunghyun Lee et al.: Validity of Propomacrus muramotoae

examined specimens were deposited in the private collection of the second
author, and the collection labels’ details are provided in Figs 1, 2.

For compatibility with public sequences, we targeted the cytochrome oxidase
subunit | (COl), previously utilized in a Propomacrus study (Sfenthourakis et al.
2017), to integrate our de novo data with the public data available on GenBank
(https://www.ncbi.nim.nih.gov/genbank/). As our samples were not in optimal
condition, we initially retrieved all available COl sequences from GenBank and de-
signed four new Propomacrus bimucronatus-specific primer sets. PCRs were per-
formed using AccuPower® PCR PreMix (Bioneer, Daejeon, Korea) and sent to BI-
ONICS Co., Ltd (Seoul, Korea) for sequencing. Public sequences used in this study,
PCR primers, and PCR conditions are described in Suppl. material 1: tables S1-S3.

We utilized MAFFT ver. 7 online (Katoh et al. 2019) for multiple sequence align-
ment, and the final alignment was visualized in GENEIOUS (Kearse et al. 2012) to
determine the position of each sequence. The amino acid translation option in
MEGA X (Kumar et al. 2018) was used for the final sequence assessment. The
phylogenetic analysis was conducted using the maximum likelihood method (ML)
with IQ-TREE (Nguyen et al. 2015). Haplotype network analysis was performed

- EPAL | DHPRAV PROV. j :
: hihi pid site |PROPOMACRUS
C kK BAK! MURAMOTORE
4 VI -S 246 -200§ FUTIOK :
et .B.C. Ratcliffe 2008

LUSGUONHYA Cocz.

= SL! MMe ei J - 2 y
Figure 1. Propomacrus muramotoae labeled as being collected from Nepal A lateral margin of pronotum, dorsolateral
view. Light green lines highlight artificial grinding B lateral margin of pronotum, dorsal view. Light green lines highlight a

punctation cut in the middle C lateral margin of pronotum of P bimucronatus D dorsal habitus E ventral habitus.

ZooKeys 1206: 181-190 (2024), DOI: 10.3897/zookeys.1206.124932 183

Seunghyun Lee et al.: Validity of Propomacrus muramotoae

using the TCS algorithm (Clement et al. 2000) implemented in PopART ver. 1.7
(Leigh and Bryant 2015). Sequences were categorized into six groups, represent-
ing each taxonomic unit (Propomacrus muramotoae, P. bimucronatus bimucrona-
tus, P bimucronatus cypriacus, P. davidis, Euchirus dupontianus and E. longimanus).

Results
Morphological examination

The three specimens labeled as “Propomacrus muramotoae” exhibited unusual
morphological features (Figs 1, 2). Firstly, R muramotoae labeled as being col-
lected in “Nepal” exhibited notably blunt lateral pronotal processes as described
in the original species description (Fig. 1D). The development of the elytral lon-

Pl sa, gy me
A =

ry ff -.
>) LIVIEQ
be ‘

Figure 2. Propomacrus muramotoae labeled as being collected from Tibet A dorsal habitus, male B ventral habitus, male
C dorsal habitus, female D ventral habitus, female E forefemur of male with black stain F lateral margin of pronotum,
dorsal view G lateral margin of pronotum, ventral view.

ZooKeys 1206: 181-190 (2024), DOI: 10.3897/zookeys.1206.124932 184

Seunghyun Lee et al.: Validity of Propomacrus muramotoae

gitudinal costa was weak, and a ventral longitudinal groove was absent. How-
ever, microscopic analysis of the blunted areas on the lateral pronotal process
indicated clear signs of artificial grinding. When observed from the side, the
edges of the areas subjected to grinding were not smooth but were instead
bluntly truncated throughout (Fig. 1A). The punctation on the plate was cut in
the middle with a straight line across the area suspected of having been ground
(Fig. 1B). The P muramotoae specimens labeled as being collected from “Tibet”
displayed a mottled black coloring across both sexes, appearing to be artificial-
ly dyed (Fig. 2A-G). Each specimen featured sharply developed lateral pronotal
processes (Fig. 2F), with the elytral longitudinal costa weakly developed and
a ventral longitudinal groove absent (Fig. 2B, D). These specimens have the
diagnostic characters of P bimucronatus, with the exception of their coloration.

Molecular analyses

In the network analysis, we identified a total of 17 haplotypes within the P. bimu-
cronatus species complex (P. b. bimucronatus + P. b. cypriacus). Within these,
Pb. cypriacus exhibited notable diversity, presenting 14 distinct haplotypes.
Conversely, only two haplotypes were observed in P. b. bimucronatus. Notably, a
predominant haplotype, designated as haplotype A, was shared by the majority
of individuals studied. This haplotype was particularly significant in our analy-
sis of RP muramotoae; all five sequences examined were identical to haplotype
A. In terms of haplogroups, P. b. cypriacus formed a distinct group, while the
remaining sequences recovered polyphyletic (Fig. 3).

Furthermore, monophyly of P bimucronatus species complex was recovered
with strong Ultrafast Bootstrap Support (UBS = 100) within the maximum like-
lihood (ML) tree. In the phylogenetic tree, P muramotoae was clearly nested
within the P bimucronatus clade. The clade, which included five P muramotoae
specimens was monophyletic with a branch length of zero and high support
values (UBS = 99). Consistent with the network analysis, the P. b. cypriacus
clade formed monophyletic groups with high supporting values (UBS = 90),
reinforcing the results observed in the haplotype analysis (Fig. 3).

Discussion

Our DNA analysis showed a variety of haplotypes of P. b. cypriacus among the ex-
tensive samples from the small island of Cyprus. Conversely, P. b. bimucronatus,
with a distribution over a significantly larger area, has a disproportionately small
number of sequences uploaded to GenBank relative to its range, with all speci-
mens collected in Turkey. Therefore, widely distributed P b. bimucronatus should
exhibit higher genetic diversity than P b. cypriacus, as a wider range correlates
with greater genetic diversity in close congeners (Cole 2003; Leffler et al. 2012;
Hague and Routman 2016). Furthermore, existing studies of the genus Cheiro-
tonus, which is closely related to Propomacrus (Yu et al. 2023), demonstrate
significant intraspecific variation in genetic diversity correlated with its species’
distribution patterns: Cheirotonus gestroi Pouillaude, 1913, which has a wide dis-
tribution, shows a broad range of genetic variation (Yang et al. 2020), in contrast
to Cheirotonus formosanus Ohaus, 1913, which has a narrower distribution and
exhibits lesser genetic variation (Huang et al. 2024). It is particularly unconvinc-

ZooKeys 1206: 181-190 (2024), DOI: 10.3897/zookeys.1206.124932 185

Seunghyun Lee et al.: Validity of Propomacrus muramotoae

KY004195.1_Euchirus_long|
KY004194.1_Euchirus_dupontianus_33
KY004193.1_Propomacrus_davidi_29
= KY004188.1_Propomacrus_bimucronatus_22
KY004187.1_Propomacrus_bimucronatus_21

KY004191.1_Propomacrus_bimucronatus_25

D1_Propomacrus_muran
D6 _Propomacrus_mura
D4_Propomacrus_murar
j D3 _Propomacrus_mur
Euchirus
O) D2_Propomacrus_muran

O NC_070352.1_Propomacrus_bimucronatus

O KY004190.1_Propomacrus_bimucronatus_24
L ropomacrus KY004189.1_Propomacrus_bimucronatus_23
bimucronatus spp. KY004192.1_Propomacrus_bimucronatus_27

KY004179.1_Propomacrus_cypriacus_13

KY004176.1_Propomacrus_cypriacus_10
KY004173.1_Propomacrus_cypriacus_7
| KY004178.1_Propomacrus_cypriacus_12

ait KY004174.1_Propomacrus_cypriacus_8
KY004185.1_Propomacrus_cypriacus_1
KY004171.1_Propomacrus_cypriacus :
'004172.1_Propomacrus_cypriacus.
KY004170.1_Propomacrus_cypriacus
KY004180.1_Propomacrus_cypriacus_1é

KY004181.1_Propomacrus_cypriacus_15
KY004182.1_Propomacrus_cypriacus_16
KY004169.1_Propomacrus_cypriacus_3
KY004184.1_Propomacrus_cypriacus_1 3
KY004168.1_Propomacrus_cypriacus_2
KY004183.1_Propomacrus_cypriacus_17
KY004167.1_Propomacrus_cypriacus_1
KY004186.1_Propomacrus_cypriacus,

awa,

KY004175.1_Propomacrus_cypriacus_S

KY004177.1_Propomacrus_cypriacus_

Figure 3. Genetic analyses using COI gene. Lower left. Haplotype network analyses. Abbreviations PM: Propomacrus mu-
ramotoae, PBT: P bimucronatus bimucronatus, PBC: P bimucronatus cypriacus, PD: P. davidis, ED: Euchirus dupontianus,
and EL: E. longimanus. Right. Phylogenetic relationships of Propomacrus resulting from IQtree. High Ultrafast bootstrap
support values (290) are marked with black circles.

ing that individuals found with the labels “Nepal” and “Tibet” possess a COI hap-
lotype identical to the most common haplotype identified in Turkey populations,
raising suspicions about the uniformity of sequences between the individuals.

The morphological characteristics of the species are also notably ambigu-
ous. The lateral pronotal process considered a distinctive feature of P mura-
motoae, was only observed in one specimen, where it appeared to have been
artificially modified. This modification is particularly prominent in a sharply cut
punctuation along the lateral margin. The presence of the elytral longitudinal
costa, a trait often found in P bimucronatus, adds to the ambiguity, along with
the absence of the abdominal longitudinal groove in all specimens examined.
The remaining two specimens, labeled as from Tibet, were indistinguishable
from P. bimucronatus in both DNA and morphological aspects and lacked any
diagnostic features of P muramotoae according to the original description.

It is essential to recognize that all specimens of P muramotoae were exclu-
sively provided by an insect dealer, Li Jingke (personal communications with
the second author). Li Jingke has a well-documented reputation as a fraudster
(Suppl. material 2: fig. S1) even though his fraudulent activities have rarely been
formally reported (Han et al. 2017). Probably, the specimens were reared from
larvae or obtained from common sources, such as bred population from Turkey,
and subsequently altered to sell at high prices. Surprisingly, in several advertis-
ing emails from Li Jingke that we received, we found descriptions of specimen

ZooKeys 1206: 181-190 (2024), DOI: 10.3897/zookeys.1206.124932 186

Seunghyun Lee et al.: Validity of Propomacrus muramotoae

variations that appeared completely random, such as white lines on the elytral
margin, wider elytra, and a bleached posterior half (Suppl. material 2: fig. S2).
Specimens advertised as having these ‘unique features’ were sold at very high
prices (Suppl. material 2: fig. S2). Such practices, though inconceivable within
the scientific community, unfortunately do exist. Direct manipulation of spec-
imens is rarely documented in entomology (Braby and Eastwood 2019). The
morphological alterations by the fraudster were carelessly executed in this
case, and fortunately, DNA barcode amplification was successful. However, it
should be noted that such verification may not always be possible.

Based on genetic and morphological analysis, coupled with indirect data dis-
cussed above, we believe that the type of P muramotoae is an altered specimen
of P bimucronatus. Therefore, we propose that P muramotoae Fujioka, 2007, is
a junior synonym of P bimucronatus Pallas, 1781. A significant limitation of our
study, however, is the absence of examination and genetic analysis of the type
specimens. The type specimens of P muramotoae are housed at the National Mu-
seum of Nature and Science in Tokyo, Japan, according to the original description.
However, we were unable to find the types for our research; they were not deposit-
ed at the National Museum of Nature and Science and it is presumed they remain
within the collection of the original describer. All authors tried to contact him in var-
ious ways but failed to access the type specimens. The lack of genetic divergence
from P. bimucronatus and clear evidence of morphological manipulation strongly
indicate that P muramotoae represents a significant taxonomic deception. Our re-
search indicates that verification of the type specimen is feasible and straightfor-
ward and we suggest those with access to the holotype conduct official taxonomic
verification of P muramotoae: simply amplify and do molecular analyses using the
COI barcode region and examine external morphology under a microscope.

Taxonomic account

Tribe Euchirini Hope 1840

Genus Propomacrus Newman, 1837

Porropus Laporte 1840: 113.
Protomacrus Hope 1841: 595.
Macropropus Agassiz 1846: 309.

Type species. Scarabaeus bimucronatus Pallas, 1781: 13.

Propomacrus bimucronatus bimucronatus Pallas, 1781

Scarabaeus bimucronatus Pallas, 1781: 13.
Propomacrus arbaces Newman, 1837: 256.
Propomacrus muramotoae Fujioka, 2007: 99. (syn. nov.)

Material examined. TURKEY * 1 male, 2 females; Mersin province 1500 m nr.
K6secobanli village dead in old pollarded oaks; 2017; Serder Goktepe leg.;
BMNH{E} 2018-74; Natural History Museum London (NHM hereafter) * 1 male;

ZooKeys 1206: 181-190 (2024), DOI: 10.3897/zookeys.1206.124932 187

Seunghyun Lee et al.: Validity of Propomacrus muramotoae

Smyrna; NHM « 1 male; Asia Minor; 1910; G.a. Tellalian; NHM + 1 female; Asia
Minor; NHM + 1 male, 2 females; Fry coll.; As Min Smyrne; 1905-100; NHM
- 1 female; Besika Bay; G.C.C. Champion; 1927-409; NHM : 3 males, 1 female;
Smyrne; 1906; Chinese Academy of Sciences * 2 males, 2 females; Hatay; Jun.
2007; private collection of Woong Choi. SYRIA * 2 females; Syria; 80.53; NHM
- 1 female; Aleppo, Syria; G Lewis; 1915-38; NHM.

Additional material with falsified labels. NEPAL 1 female; Knandbari, Dharan
Prov.; 5 Jun. 2008; LUOGUOHUA leg.; private collection of Seulmaro Hwang;
CHINA 1 male, 1 female; Madi (Medog) county, Linzhi (Nyingchi), Xizang;
29°29'N, 95°30'E; alt. c. 600 m; 7-15 Jul. 2014; Wu Weimin leg.; private collec-
tion of Seulmaro Hwang.

Propomacrus bimucronatus cypriacus Alexis & Makris, 2002
Propomacrus cypriacus Alexis & Makris, 2002: 103.

Material examined. Cyprus: 1 male; Alethriko, Larnaca; 34°51.54'N, 33°29.38'E;
15. viii. 2006; Aristos Aristophanous leg.; BMNH{E}2015-88; NHM «+ 1 female
Alethriko, Larnaca; 34°51.54'N, 33°29.38'E; 5 ix 2008; Aristos Aristophanous
leg.; BMNH{E}201 5-88; NHM.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by Basic Science Research Program through the Nation-
al Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-
2023-00237795); National Key R&D Program of China (Nos. 2022YFC2601200,
2023YFC2604904); the Survey of Wildlife Resources in Key Areas of Tibet (ZL202203601);
and the National Science & Technology Fundamental Resources Investigation Program
of China (Nos. 2023FY100301, 2022FY100500).

Author contributions

Conceptualization: SL. Data curation: SL, WC, ML, JS, SH. Formal analysis: SL. Funding
acquisition: MB, SL. Investigation: WC, SH, JS, ML. Resources: SH, MB, WC. Supervision:
MB. Visualization: SL. Writing - original draft: SL. Writing - review and editing: MB, SH,
JS, ML, WC, SL.

Author ORCIDs

Seunghyun Lee © https://orcid.org/0000-0001-6318-4116
Minhyeuk Lee © https://orcid.org/0000-0002-5667-9097
Jinbae Seung ® https://orcid.org/0000-0001-91 15-1733
Ming Bai © https://orcid.org/0000-0001-9197-5900

ZooKeys 1206: 181-190 (2024), DOI: 10.3897/zookeys.1206.124932 188

Seunghyun Lee et al.: Validity of Propomacrus muramotoae

Data availability

All of the data that support the findings of this study are available in the main text or
Supplementary Information.

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Supplementary material 1

Public sequences, PCR primers, and PCR conditions

Authors: Seunghyun Lee, Seulmaro Hwang, Minhyeuk Lee, Jinbae Seung, Woong Choi,
Ming Bai

Data type: xlsx

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/zookeys.1206.124932.suppl1

Supplementary material 2

Deceptive practices of Li Jingke and sales email from him

Authors: Seunghyun Lee, Seulmaro Hwang, Minhyeuk Lee, Jinbae Seung, Woong Choi,
Ming Bai

Data type: pdf

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/zookeys.1206.124932.suppl2

ZooKeys 1206: 181-190 (2024), DOI: 10.3897/zookeys.1206.124932 190