Exploring the diversity of Eupolyphaga Chopard, 1929 (Blattodea, Corydioidea): species delimitation based on morphology and molecular analysis

Abstract

Eupolyphaga Chopard, 1929 is a cockroach genus mainly endemic to China. In this study, the species diversity of this genus is further explored through morphology and molecular analysis. Four species are described new to science: Eupolyphaga miracidia Qiu, sp. nov. , Eupolyphaga udenostyla Qiu, sp. nov. , Eupolyphaga reducta Qiu, sp. nov. , and Eupolyphaga simila Qiu, sp. nov. New knowledge on some known species is added, including new distribution records and characteristics of females. Forty-five COI sequences were newly sequenced and a molecular species delimitation analysis was performed using ABGD method. Eighteen molecular operational taxonomic units were obtained by ABGD analysis, which are nearly consistent with the results of morphological delimitation.

Full text

Exploring the diversity of Eupolyphaga Chopard, 1929
(Blattodea, Corydioidea): species delimitation
based on morphology and molecular analysis
Wei Han1, Lu Qiu1,2, Jing Zhu1, Zong-Qing Wang1, Yan-Li Che1
1 Institute of Entomology, College of Plant Protection, Southwest University, Beibei, Chongqing 400716,
China 2Engineering Research Center for Forest and Grassland Disaster Prevention and Reduction, Mianyang
Normal University, Mianyang 621000, China
Corresponding author: Yan-Li Che (lilyche@swu.edu.cn)
Academic editor: Fred Legendre|Received 8 June 2022|Accepted 22 July 2022|Published 5 September 2022
https://zoobank.org/4B7BD929-48A9-4517-8DF2-7F6780BC1AD3
Citation: Han W, Qiu L, Zhu J, Wang Z-Q, Che Y-L (2022) Exploring the diversity of Eupolyphaga Chopard, 1929
(Blattodea, Corydioidea): species delimitation based on morphology and molecular analysis. ZooKeys 1120: 67–94.
https://doi.org/10.3897/zookeys.1120.87483
Abstract
Eupolyphaga Chopard, 1929 is a cockroach genus mainly endemic to China. In this study, the species
diversity of this genus is further explored through morphology and molecular analysis. Four species are
described new to science: Eupolyphaga miracidia Qiu, sp. nov., Eupolyphaga udenostyla Qiu, sp. nov.,
Eupolyphaga reducta Qiu, sp. nov., and Eupolyphaga simila Qiu, sp. nov. New knowledge on some known
species is added, including new distribution records and characteristics of females. Forty-ve COI se-
quences were newly sequenced and a molecular species delimitation analysis was performed using ABGD
method. Eighteen molecular operational taxonomic units were obtained by ABGD analysis, which are
nearly consistent with the results of morphological delimitation.
Keywords
ABGD, Corydiinae, DNA barcoding, new species, Polyphagini
Introduction
Eupolyphaga Chopard, 1929 is the largest genus of Corydioidea in China, including 20
species and two subspecies (Chopard 1929; Feng and Woo 1988; Woo and Feng 1992;
Qiu et al. 2018). Sexual dimorphism is pronounced in Eupolyphaga species: the male adult
ZooKeys 1120: 67–94 (2022)
doi: 10.3897/zookeys.1120.87483
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Wei Han et al. / ZooKeys 1120: 67–94 (2022)
68
is winged with its body narrowly oval, while the female adult is apterous with a sub-oval
body. Although this genus is famous for having species with medicinal value, e.g., E. sinensis
farmed in China as one kind of traditional Chinese medicine (Woo 1987), the taxonomic
history of Eupolyphaga is rather brief. is genus was established by Chopard (1929) with
Eupolyphaga sinensis as the type species, and a total of ve species was included. After this,
an additional two species from Yunnan were described (Feng and Woo 1988; Woo and
Feng 1992). ere was no further taxonomic work on this genus until Qiu et al. (2018),
who made a revision to this genus and updated the total number of species to 20.
Species delimitation of the genus has been based on external morphological char-
acters, male genitalia and some oothecae characters (Chopard 1929; Feng and Woo
1988; Woo and Feng 1992; Qiu et al. 2018). Qiu et al. (2018) pointed out that male
genitalia played a limited role in species identication of Eupolyphaga, because most
species do not show very clearly distinct features. As more specimens are collected,
boundaries between some species become ambiguous. For example, a new species de-
scribed below (SWU-B-CC-010023 to SWU-B-CC-010027) collected in Sichuan
province is very similar to E. yunnanensis in males, while their females and oothecae
have signicant dierences, we therefore consider them to belong to dierent species.
Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012) is now a com-
mon DNA-based delimitation method. So far, it has been successfully applied in many
studies about cockroaches (e.g., Bai et al. 2018; Yang et al. 2019; Deng et al. 2020;
He et al. 2021; Wang et al. 2021; Zhu et al. 2022), and this method combined with
morphological characters can help us judge whether they are dierent species or not.
We recently obtained more Eupolyphaga material from various collecting sites in
China. In this study, we use morphological characteristics combined with ABGD to
delimit species. Four new species are established. Some descriptions of females and oo-
thecae of known species, and some new distributional data are added to complement
species descriptions and facilitate future identications.
Materials and methods
Material
Specimens, including the voucher specimens (Table 1) studied in this article, are de-
posited at the Institute of Entomology, College of Plant Protection, Southwest Univer-
sity, Chongqing, China (SWU).
Morphology
e morphological terminology follows Roth (2003), Klass (1997) and Li et al. (2018).
Male genital segments of the examined specimens were macerated and heated in 10%
NaOH solution for ~ 20 min to remove excess fat, then washed with deionized water,
placed in glycerin jelly, and observed with a Motic K400 or Leica® M205A stereomi-

Species delimitation based on morphology and molecular analysis 69
croscope. Habitus photographs of the specimens were taken using a Canon® EOS M5
digital camera+ mount adapter EF-EOS M plus a Laowa 100 mm F2.8 CA-Dreamer
Macro 2 × lens (for Canon EF). Photographs of other characters were taken using a
Leica® M205A stereomicroscope. All photographs mentioned above were optimized
with Adobe Photoshop® CC 2019.
Table 1. Samples used in species delimitation.
Species Abbreviation GenBank ID Collecting information Remark
E. sinensis EupoSineLN OP215845 Tongnai Village, Fuxin, Liaoning; 31 July 2016; Lu-
YuWang
nymph
EupoSineBJ OP215846 Mt. Xishan, Beijing; 28 April 2015; Bing-Qiang Wang male
EupoSineWH OP215847 Sushansi, Wuhan, Hubei; 16 August 2019; Chen-Liang Li male
E. hanae EupoHanaSM OP215848 Mt. Simianshan, Jiangjin, Chongqing; 31 August 2018;
Lu Qiu
male
EupoHanaDG OP215849 Daguan Town, Dujiangyan, Sichuan; 19 May 2015; Lu
Qiu, Jing-Fei Han
male
EupoHanaSN1 OP215850 Shehong, Suining, Sichuan; 8 May 2016; Lei Wang male
EupoHanaSN2 OP215851 ibid female
EupoHanaJY OP215852 Mt. Jinyunshan, Beibei, Chongqing; 22 August 2018;
Lu Qiu
male
EupoHanaGL OP215853 Jianzhu Township, Gulin, Sichuan; 1 February 2019; Lu Qiu female
E. hupingensis EupoHupiSM OP215854 Hupingshan Town, Shimen, Hunan; 21 May 2016;
HaoXu
male
EupoHupiJZ OP215855 Jingzhai, Lu,an, Anhui; 2 August 2018; Yu-chen Zheng male
E. robusta EupoRobu1 OP215856 Maoxian, Sichuan; 6 August 2019; Lu Qiu, Wei Han,
Huan-Yu Ren
male, orange
abdominal type
EupoRobu2 OP215857 ibid female
EupoRobu3 OP215858 Miancu Village, Maoxian, Sichuan; 7 August 2019; Zong-
Qing Wang
male, orange
abdominal type
EupoRobu4 OP215859 Miancu Village, Maoxian, Sichuan; 7 August 2019; Wei
Han, Huanyu Ren
female
EupoRobu5 OP215860 Wenchuan, Sichuan; 7 August 2019; Wei Han,
HuanyuRen
male, orange
abdominal type
EupoRobu6 OP215861 Wenchuan, Sichuan; 7 August 2019; Zongqing Wang female
EupoRobu8 OP215862 Wenchuan, Sichuan; 7 August 2019; Wei Han, Huan-
YuRen
male, black
abdominal type
EupoRobu7 OP215863 Miansi Town, Wenchuan, Sichuan; 29 May 2020; Jian-
Yue Qiu
male, black
abdominal type
E. yunnanensis EupoYunnCB OP215864 Zayü, Tibet; 1 August 2014; Weiwei Zhang female
EupoYunnCY OP215865 Zayü, Tibet; 14 August 2015; Lu Qiu male
EupoYunnBO OP215866 Bomê, Tibet; 11 July 2016; Jian-Yue Qiu, Hao Xu male
EupoYunnTM2 OP215867 Tongmai Town, Bomê, Tibet; 13 August 2017; HaoXu,
Jian-Yue Qiu
male
EupoYunnTM3 OP215868 Tongmai Town, Bomê, Tibet; 11 August 2017; Hao Xu,
Jian-Yue Qiu
male
EupoYunnTM1 OP215869 Tongmai Town, Bomê, Tibet; 12 August 2017; Jian-Yue
Qiu, Hao Xu
male
E. fengi EupoFeng1 OP215870 Mt. Zixishan, Chuxiong, Yunnan; 7 July 2012;
DongWang
male
EupoFeng2 OP215871 ibid male
E. dongi EupoDong1 OP215872 Mt. Gaoligongshan, Baoshan, Yunnan; 13 April 2017;
Zhi-Wei Dong
male
EupoDong2 OP215873 Mt. Gaoligongshan, Baoshan, Yunnan; June 2020; Lu
Qiu, Jin-Lin Liu
nymph
E. wooi EupoWooi OP215874 Mt. Ailaoshan, Xinping, Yunnan; 11 May 2016; Lu Qiu female

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
70
DNA extraction, PCR, and sequencing
e hind legs or thoracic muscle were used for DNA extraction, and the other body
parts were stored in 95% ethanol as voucher specimens. e extraction procedure
was performed according to the Hipure Tissue DNA Mini Kit (Magen Bio-tech,
Guangzhou) and the extracted total DNA was stored in a -20 °C environment. Frag-
ments of COI were amplied using PCR; primers used for the amplications are
F: 5’-GGTCAACAAATCATAAAGATATTGG-3’ and R: 5’-TAAACTTCAGGGT-
GACCAAAAAATCA-3’ (Folmer et al. 1994). PCR was performed in an Analytik
Jena Easy Cycler with 25 µl volumes including 22 µL 3×Taq Master Mix, 1 µL of each
primer (F and R, aforementioned), and 1 µL of DNA template. Amplication condi-
tions were: initial denaturation at 98 °C for 2 min, followed by 35 cycles for 10 s at
98 °C, 10 s at 50 °C, 10 s at 72 °C, and a nal extension of 5 min at 72 °C. en the
amplication eect was assessed by electrophoresis in 1% agarose gel, the amplica-
tion products corresponding to bright and clear bands were selected and sent to the
Tsingke Biotechnology Co., Ltd. (Beijing, China) for sequencing in both directions.
Sequence processing and phylogenetic analyses
In total, 48 COI sequences were used for analysis (45 sequences representing
Eupolyphaga species and three sequences representing outgroups downloaded from
Species Abbreviation GenBank ID Collecting information Remark
E. xuorum EupoXuor1 OP215875 Caoke Township, Shimian, Sichuan; 25 August 2016;
Hao Xu, Jian-Yue Qiu
male
EupoXuor2 OP215876 ibid male
E. daweishana EupoDawe OP215877 Mt. Daweishan, Pinbian, Yunnan; 16 May 2016; Lu Qiu nymph
E. miracidia sp. nov. EupoMira OP215878 Maqiao Town, Xiangyang, Hubei; 13 July 2017; LuQiu male
E. nigrinotum EupoNigr1 OP215879 Mt. Jizushan, Bingchuan, Yunnan; 20 February 2016;
Hao Xu, Jian-Yue Qiu
male
EupoNigr3 OP215880 Mt. Jizushan, Bingchuan, Yunnan; 20 February 2016;
Hao Xu, Jian-Yue Qiu
female
EupoNigr2 OP215881 Mt. Jizushan, Bingchuan, Yunnan; 7 June 2019, local male
E. pilosa EupoPilo OP215882 Pantiange Township, Weixi, Yunnan; 21 August 2015;
Lu Qiu
male
E. simila sp. nov. EupoSimi1 OP215883 Miyaluo Town, Lixian, Sichuan; 6 October 2019; Lu Qiu,
Hao Xu, Zhi-Teng Chen
female
EupoSimi2 OP215884 ibid nymph
EupoSimi3 OP215885 ibid male
E. reducta sp. nov. EupoRedu OP215886 Wadi Township, Maoxian, Sichuan; 3 October 2019; Hao
Xu, Zhi-Teng Chen, Lu Qiu
nymph
E. udenostyla sp. nov. EupoUden1 OP215887 Keku Township, Wenchuan, Sichuan; 7 August 2019;
Huan-Yu Ren, Wei Han
female
EupoUden2 OP215888 ibid male
EupoUden3 OP215889 ibid male
Outgroup
Periplaneta americana PeriAmer HM386405 /
Eucorydia dasytoides EucuDasy LC480880 /
Diploptera punctata DiplPunc MF479156 /

Species delimitation based on morphology and molecular analysis 71
GenBank). All 45 newly acquired sequences are deposited in GenBank (https://
www.ncbi.nlm.nih.gov/nuccore) with accession numbers OP215845 to OP215889
(Table 1). Sequences were aligned by online MAFFT 7 (https://mat.cbrc.jp/
alignment/server/) (Katoh et al. 2019) using the Q-INS-i algorithm, then aligned
and adjusted manually after translation into amino acid sequences using MEGA
11 (Kumar et al. 2016). A Kimura 2-parameter (Kimura 1980) distance model
was used to quantify intraspecic and interspecic genetic divergence values. e
maximum likelihood tree was constructed in IQ-TREE (Nguyen et al. 2015) with
1000 replicates for bootstrap values, after choosing optimal partitioning scheme
and substitution models (COI_pos 1, TRN+G; COI_pos 2, GTR+G; COI_pos 3,
GTR+G) in PartionFinder v.2.1.1 (Lanfear et al. 2017) with the corrected Akaike
Information Criterion (AICc).
We also conducted an ABGD analysis to confront its results to our morpho-
logically dened species and thus further rene our species delimitation within
the genus Eupolyphaga. ABGD analysis was performed using a web interface
(https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html); the default parameters
were used except for the relative gap width set at 1.0 and using the Jukes-Cantor
(JC69)model.
Results
Morphological delimitation of Eupolyphaga
Combining male and female external morphology, male genitalia, and some oothecae
features, we identied 16 morphospecies (including four new species) of Eupolyphaga
among 266 samples examined from China (Fig. 1A).
Phylogenetic analysis and MOTUs estimation based on COI
In this study, we acquired 45 COI sequences of Eupolyphaga representing 16 morphos-
pecies of Eupolyphaga. e maximum likelihood tree shows that, for each morphologi-
cal species, all samples (including the dierent locality samples, dierent sex samples,
and adult and nymphal samples) are monophyletic, although most of the nodes do not
have high bootstrap values (Fig. 1).
Eighteen molecular operational taxonomic units (MOTUs) were estimated by
ABGD for the 45 samples (Fig. 1B), and 14 morphological species were well sup-
ported by the ABGD result; morphological assumptions and molecular results dier
only for E. sinensis and E. hupingensis. e three samples of E. sinensis collected from
three dierent localities (Beijing, Liaoning, and Wuhan) are estimated as two MOTUs.
e two samples of E. hupingensis, collected from Hunan and Anhui provinces, were
also divided into two MOTUs.

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
72
Figure 1. Maximum Likelihood tree derived from COI sequences with 1000 bootstrap replicates. Boot-
strap values are marked at nodes, the sexes of the specimens are given in brackets (“N” indicates nymphs).
Red bar: results of morphological delimitation; blue bar: results of MOTUs estimated by ABGD.

Species delimitation based on morphology and molecular analysis 73
Systematics
Eupolyphaga miracidia Qiu, sp. nov.
https://zoobank.org/90674A34-2596-438B-9668-5CEAA24103EE
Fig. 2
Type material. Holotype: C · male; Hubei Prov., Xiangyang City, Maqiao Town,
roadside of Ganxigou; 480 m–600 m; 31°46.99'N, 110°55.06'E; 13 July 2017; Lu
Qiu leg.; SWU-B-CC-010001.
Paratypes: C · 2 males & 2 females, same collection data as holotype; SWU-
B-CC-010002 to SWU-B-CC-010005.
Diagnosis. is species can be easily distinguished from other species by its small
body size. It resembles E. hupingensis by its dark coloration and dense maculae on teg-
mina, but it can be distinguished from the latter by its small-sized body in both sexes
(12.1–12.5 mm excluding tegmina and wings in males, 22.3–24.1 mm in females;
Fig. 2A–D), small styli (Fig. 2J), slenderer genital hook (Fig. 2K), and the relatively
separated spaces between the serrations of the ootheca (Fig. 2M, N).
Description. Male holotype. Measurements (mm). Overall length: 26.9;
body length: 12.1; body width (tegmina not included): 9.8; tegmen length × width:
22.3×9.5; pronotum length × width: 7.4 × 4.4.
Coloration. Body blackish brown, covered with brown setae (Fig. 2A, B). Head
black, ante-clypeus whitish, sub-transparent. Antenna brown. Pronotum dark-brown,
anterior margin yellowish white. Tegmina sub-transparent, yellowish brown, with dark
brown maculae. Wing light brown, with darkish brown maculae. Leg blackish brown,
with coxa and trochanter slightly tawny. Abdomen blackish brown, slightly yellowish
brown medially.
Head: Subrounded, almost hidden under pronotum. Interocular space moder-
ate, about half of the distance between ocelli. Ocelli large, ocelli ridge protruded and
curved, two dimples symmetrically situated below the ridge. Two yellowish brown
semicircular pits each situated next to the inner side of antennal sockets. Clypeus
distinct, labrum almost quadrate, posterior margin concave in the middle (Fig. 2F).
Pronotum: Small, widest near the middle, anterior whitish margin narrow, gradually
narrowing from the middle to lateral sides, boundary between the white and dark
brown areas distinct (Fig. 2E). Tegmina and hind wings: Extending beyond the end
of abdomen 12.1 mm, maculae on tegmina dense, fused. Legs: Slender, front femur
type C1, pulvilli moderate and tarsal claws simple, symmetrical, arolia large. Abdomen:
Supra-anal plate transverse, pubescent, posterior margin prominent medially, parap-
rocts simple, cerci short (Fig. 2I). Subgenital plate asymmetrical, hind margin concave
medially and densely setose, left side less prominent than right side. Styli small, the
left one longer than the right one (Fig. 2J). Genitalia: L1 basally prolonged (Fig. 2K),
genital hook (L3) slender, the hooked part roundly curved, apex sharp (Fig. 2K). Right
phallomere small, R2 simple, broad, and concave in the middle (Fig. 2L).
Male paratypes. Similar to the holotype, no distinct dierences.

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
74
Figure 2. Eupolyphaga miracidia Qiu, sp. nov. A male holotype, dorsal view B male holotype, ventral
view C female paratype, dorsal view D female paratype, ventral view E pronotum, male holotype, dorsal
view F head, male holotype, ventral view G pronotum, female paratype, dorsal view H head, female para-
type, ventral view I supra-anal plate, male holotype, ventral view J subgenital plate, male holotype, ventral
view K genitalia, male holotype, dorsal view L right phallomere, holotype, right-ventral view Mthe ser-
ration of oothecae N oothecae, lateral view O–R habitats of E. miracidia Qiu, sp. nov., in Maqiao Town,
Xiangyang City, Hubei Province S living male T living female. Scale bars: 1.0 cm (A–D); 0.1 cm (E–L,
N); 0.02 cm (M). Photographs O–T by LQ.

Species delimitation based on morphology and molecular analysis 75
Female paratypes. Body length: 22.3–24.1 mm, body width: 16.2–16.9 mm. Body
uniformly dark reddish brown (Fig. 2C, D). Antennal sockets and ocelli pale yellow,
ante-clypeus yellow. Labrum yellowish white, base and ends slightly lighter in color.
Ocelli moderate in size, nearly triangular. Interocular space almost equal to distance
between antennal sockets, and bigger than the distance between ocelli (Fig.2H). Arolia
and pullivi absent. Posterior margin of the supra-anal plate emarginated medially.
Nymph. Similar to the female, body darkish brown.
Ootheca. Reddish brown. e longitudinal lines distinct. Serrations on the keel
large and curved, apex of serrations slightly truncated. Space between the serrations of
the curved portion moderate (Fig. 2M). Respiratory canals well developed.
Natural history. Found in dry earth under a cli of the roadside (Fig. 2O–R).
Etymology. e species epithet miracidia is derived from Greek mirakos referring
to it's small size.
Eupolyphaga udenostyla Qiu, sp. nov.
https://zoobank.org/ED205416-E3D8-4955-AE89-7D50DEB363DE
Figs 3, 4B, D, E–J
Type material. Holotype: C · male; Sichuan Prov., Aba Prefecture, Wenchuan
County, Keku Township; 1555 m; 31°30.93'N, 103°34.27'E, 5 May 2019; Lu Qiu
leg.; SWU-B-CC-010006.
Paratypes: C · 3 males & 1 female, same collection data as holotype; SWU-B-
CC-010007 to 010010 · 3 males, same collection data as holotype, but 7 August 2019;
Huan-Yu Ren, Wei Han leg; SWU-B-CC-010011 to 010013 · 2 males & 4 females,
Sichuan Prov., Wenchuan County, mountains behind the 5·12 Wenchuan Earthquake
Memorial Museum; July–August 2019; Qi Li leg.; SWU-B-CC-010014 to 010019.
Diagnosis. is species is remarkable for the absence of styli and the short teg-
mina and strongly reduced anal elds of hind wings in male, which can easily distin-
guish the males of E. udenostyla from all other species. e serrations of ootheca are
distinctly reduced.
Description. Male holotype: Body stout. Measurements (mm). Overall length:
23.5; body length: 19.7; body width (tegmina not included): 12.1; pronotum
length×width: 17.1 × 9.4; tegmina length × width: 17.6 × 8.4.
Coloration. Body almost black, covered with black setae (Fig. 3A, B). Anterior
margin of pronotum partially white (Fig. 3A).
Head: Ovoid, longer than wide. Vertex concealed under pronotum. Eyes small, slight-
ly reduced. Interocular space wide but narrower than the distances between antennal sock-
ets and between ocelli. Ocelli moderate, ocellar ridge distinct, with a row of setae on the
upper edge (Fig. 3E). Antennae long, approximately equal to the length of the body (ver-
tex to abdominal tip) (Fig. 4G). Ocellar ridge with two large pits below. Clypeus distinct,
ante-clypeus almost quadrate, hind lateral angles obtusely rounded, posterior margin thin
in the middle and slightly concave. Pronotum: Narrow, widest near the middle, anterior

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
76
Figure 3. Eupolyphaga udenostyla Qiu, sp. nov. A male holotype, dorsal view B male holotype, ventral
view C female paratype, dorsal view D female paratype, ventral view E male head, ventral view F tegmen,
ventral view G hind wing, ventral view H supra-anal plate, ventral view I subgenital plate, ventral view
Jleft portion of subgenital plate, dorsal view K genitalia, dorsal view L right phallomere, right-ventral
view. Scale bars: 1.0 cm (A–D); 0.2 cm E; 0.5 cm (F, G); 0.1 cm (H, I , K , L ); 0.02 cm (J). Black arrows
indicate the strongly reduced anal eld and absence of styli.

Species delimitation based on morphology and molecular analysis 77
whitish margin narrow and short. Tegmina and hind wings: Shortened, only beyond the
end of abdomen 3.8 mm. Apices of wings slightly exceed tegmina in resting position. Teg-
men rounded apically (Fig. 3F). Hind wing short, oval, bluntly rounded apically, anal eld
Figure 4. A–D Characteristics and habitats of Eupolyphaga species from Minjiang River Basin A head
of female E. robusta, ventral view B head of female Eupolyphaga udenostyla Qiu, sp. nov., ventral view
Csupra-anal plate of female E. robusta, dorsal view D supra-anal plate of female E. udenostyla Qiu, sp.
nov., dorsal view E ootheca of E. udenostyla Qiu, sp. nov., lateral view F same, close-up view to show the
serration G a living male E. udenostyla Qiu, sp. nov. H a living female E. udenostyla Qiu, sp. nov. I, J habi-
tats of E. udenostyla Qiu, sp. nov., in Keku Township, Wenchuan County, Sichuan Province. Scale bars:
0.2cm (E); 0.1 cm (F). Photographs G–J by LQ. White arrows indicate the ocelli.

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
78
strongly reduced (Fig. 3G). Legs: Slender, front femur type C1. Pulvilli moderate, tarsal
claws simple and symmetrical, arolia large. Abdomen: Supra-anal plate narrowly trans-
verse, pubescent, hind margin with an emargination medially, paraprocts simple, cerci
short (Fig. 3H). Subgenital plate simple, posterior margin nearly straight, densely setose.
Styli absent (Fig. 3I, J). Genitalia: L1 narrowed at base, genital hook (L3) stout basally,
gradually tapered at distal half, the hook roundly curved, apex sharp (Fig. 3K).
Male paratypes: Similar to the holotype, no signicant variation.
Female paratypes: Body length: 23.5–24.7 mm, body width: 17.5–18.1 mm.
Body uniformly dark brown (Fig. 3C, D), ante-clypeus and base of labrum white.
Eyes and ocelli small, with a pair of large dimples between antennal sockets (Fig. 3D).
Distance between ocelli smaller than distance antennal sockets, and both smaller than
interocular space (Fig.4B). Arolia and pulvilli absent. Supra-anal plate straight at pos-
terior margin, lateral portions somewhat angular, posterior margin with a wide hollow
medially (Fig. 4D).
Nymph. Similar to the female, body uniformly black or dark brown.
Ootheca. Reddish brown. Serrations of keel small, blunt. Surface with elevated
longitudinal lines, the width of ridge wider near the serrations (Fig. 4E, F).
Natural history. is new species is distributed in the Minjiang River Basin (Wen-
chuan County) (Fig. 4I, J). Individuals hide in the soil and are active during night
(LQ, pers. obs.).
Etymology. uden- (Greek) + styla (Greek) indicate that the males of the new spe-
cies have no styli.
Remarks. e absence of styli in males makes this new species morphologically
remarkable in Eupolyphaga. Males of the species also have distinctly shortened tegmina
and wings, and extremely reduced anal areas on hind wings. e ootheca has reduced
serrations. e type locality of the new species is close to the one of Eupolyphaga
robusta, they are sympatric, distributed in Minjiang River Basin. Males and ootheca of
this new species are easily distinguished from E. robusta, but females may be confused
because of their similar black and large body. However, females of this new species have
reduced ocelli (Fig. 4B), which are obviously smaller than those of E. robusta (Fig.4A).
Also, the posterior margin of the supra-anal plate (Fig. 4D) in this new species is
obviously straighter than that of E. robusta (Fig. 4C, D).
Eupolyphaga reducta Qiu, sp. nov.
https://zoobank.org/8749380B-3F77-4033-90D3-69A43F905ADD
Fig. 5
Type material. Holotype: C · male, Sichuan Prov., Maoxian County, Wadi Township,
Shahuzhai Village; 1938 m; 31°53.20'N, 103°28.62'E; 3 October 2019; Hao Xu, Zhi-Teng
Chen, Lu Qiu leg (the holotype was reared from nymph by LQ).; SWU-B-CC-010020.
Other specimens examined. 1 male (an incompletely eclosed individual, unsuit-
able to be designated as a type specimen); SWU-B-CC-010021 · 1 female nymph,
same collection data as holotype; SWU-B-CC-010022.

Species delimitation based on morphology and molecular analysis 79
Diagnosis. is species is easily distinguished from its congeners by the reduced
and stout tegmina and hind wings in male. In comparison with other species also
possessing reduced tegmina and hind wings in males (e.g., E. everestiana reni and
E.udenostyla Qiu, sp. nov.), this new species is obviously larger (24.7 mm without
tegmina and wings in length), the tegmina with sparse macula only on distal half;
while E. everestiana reni is small in size (18.0–18.5 mm without tegmina and hind
wings in length) and with evenly distributed maculae on tegmina. E. udenostyla Qiu,
sp. nov. (19.7 mm without tegmina and wings in length) is an almost unicolored black
species with no styli in males.
Description. Male holotype: Body short and broad (Fig. 5A, B). Measurements
(mm): Overall length: 29.5; body length: 24.7; body width (tegmina not included):
13.4; pronotum length × width: 12.5 × 6.9; tegmina length × width: 22.5 × 9.3.
Coloration. Body yellowish brown. Pronotum brown, anterior margin yellow
(Fig.5C). Space between vertex to the ocelli black, face fulvous with blackish brown
spots. Antennae yellowish brown, ante-clypeus yellow, hind-clypeus brown. Labial pal-
pi and maxillary palpi yellowish brown (Fig. 5D). Tegmina and hind wings yellowish
brown, maculae brown. Legs and abdomen brown.
Head: Interocular space narrow, obviously narrower than the distance between
antennal sockets, and slightly narrower than the distance between ocelli. Ocelli
large, ocellar ridge short, straight, covered with yellowish brown setae. Clypeus
small (Fig.5D). Pronotum: Widely transverse, the widest point near the mid-
dle, anterior yellowish margin wide, the boundary between the yellowish and the
brownish portion fused. Surface with short pubescence (Fig. 5C). Tegmina and
hind wings: Reduced and robust, exceeding the end of abdomen 4.4 mm. e
apices of hind wings exceed tegmina obviously when at resting position (Fig. 5I).
Tegmina translucent, yellowish brown, irregularly with several maculae apically,
shoulders of tegmina wide, margins rounded. Wings translucent, light yellowish
brown, almost no maculae. Legs: Slender, front femur type C1, arolia large, tar-
sal claws simple and symmetrical. Abdomen: Supra-anal plate transversely broad,
convex in the middle, cerci short (Fig.5E). Subgenital plate slightly asymmetrical,
right portion larger than left, styli short (Fig.5F). Genitalia: Well sclerotized
(Fig.5G, H). L1 slender, anteriorly prominently protruded, with a long protuber-
ance on the left side and two well-developed protrusions on the posterior margin,
L2 more developed on the right side, L3 curved, with a more rounded curvature,
processa distale (pda) and processa apicale (paa) well developed, long and sturdy.
Right phallomere relatively larger, R2 divided into two chunks, outer chunk larger
than the inner one.
Female and ootheca: Unknown.
Nymph: Similar to the male in color, yellowish brown.
Natural history. is new species is distributed in the Heishui River Basin (Maox-
ian County to Heishui County). Nymphs of this new species live beneath the withered
grass at the edge of a soil slope (Hao Xu, Lu Qiu, pers. obs.; Fig. 5K, L).
Etymology. e species epithet is from the Latin reductus indicating its reduced
tegmina and wings.

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
80
Figure 5. Eupolyphaga reducta Qiu, sp. nov., male holotype A habitus, dorsal view B habitus, ventral
view C pronotum, male holotype, dorsal view D head, male holotype, ventral view E supra-anal plate,
ventral view F subgenital plate, ventral view G genitalia, dorsal view H right phallomere, right-ventral
view I–J the living holotype after moulting K habitat of E. reducta Qiu, sp. nov., in Shahuzhai Village,
Maoxian County, Sichuan Province L Dr. Zhi-Teng Chen and Dr. Hao Xu collecting this species. Scale
bars: 1.0cm (A, B); 0.2 cm (C , D); 0.1 cm (E–H). Photographs I–L by LQ.

Species delimitation based on morphology and molecular analysis 81
Eupolyphaga simila Qiu, sp. nov.
https://zoobank.org/903D40EF-A9B2-49A7-9296-A5749B3D6D9D
Figs 6, 7
Type material. Holotype: C · male; Sichuan Prov., Lixian County, Miyaluo
Town, Siboguo Village; 2944 m; 31°41.58'N, 102°44.80'E; 6 October 2019; Hao Xu,
Lu Qiu, Zhi-Teng Chen leg.; SWU-B-CC-010023.
Paratypes: 2 males, same collection data as holotype; SWU-B-CC-010024 to 010025.
Other material examined. 2 nymphs & 4 oothecae, same collection data as holo-
type; SWU-B-CC-010026 to 010027.
Diagnosis. e male of this new species resembles Eupolyphaga yunnanensis in ex-
ternal morphology. However, the male of E. simila Qiu sp. nov. has a darker abdomen
(Fig. 6B) than E. yunnanensis. e female and nymph of this new species have dense
black markings on their bodies (Fig. 7G, H), while E. yunnanensis is without black
markings. e serrations of ootheca are bluntly rounded in this new species (Fig. 7A,
B) while they are triangular in E. yunnanensis (Qiu et al. 2018).
Description. Male holotype: Measurements (mm). Overall length: 28.9; body
length: 16.2; body width (tegmina not included): 9.4; pronotum length × width:
8.3×5.1; tegmina length × width: 26.3 × 9.0.
Coloration. Body fulvous (Fig. 6A, B). Vertex to the space between ocelli blackish
brown, ocellar ridge slightly lighter in color, the rest of the face yellow. Antennae and
labrum yellow-brown (Fig. 6D). Pronotum brown, except for the yellow anterior mar-
gin (Fig. 6C). Tegmina fawn with scattered and unequal-sized maculae. Wings nearly
transparent, with sparsely small maculae terminally. Legs fulvous. Abdomen darkish
brown, with a yellow vertical line in the middle (Fig. 6A, B).
Head. Round, longer than wide. Interocular space narrower than the distances
between antennal sockets and ocelli. Ocelli bulged, ocellar ridge pubescent (Fig. 6D).
Pronotum. Transversely elliptical, with anterior margin raised medially, and slightly
truncated, the widest point located near the middle, and the posterior margin slightly
arched. Boundary between anterior yellowish margin and the brownish portion fused
(Fig. 6C). Tegmina and hind wings. Tegmina slender, beyond the end of abdomen
13 mm. Wings transparent. Legs. Slender and hairy, arolia large. Abdomen. Posterior
margin of supra-anal plate protruded posteriorly, with a median shallow concavity
(Fig. 6E). Subgenital plate slightly asymmetrical, media widely concaved, styli long
(Fig. 6F). Genitalia. Genital hook (L3) roundly curved, with a sharp apex. R2 divided
into two asymmetrical chunks, slightly quadrate (Fig. 6G, H).
Male paratype: Body length 29.1 mm including tegmina and hind wings, no
signicant dierences from the holotype.
Nymph. Yellowish brown, dorsal surface black-brown to pale yellow-brown, with
many black maculae. Vertex with a black strip between eyes, the space between ocelli
with some black spots. Hind-clypeus black. Legs darkish brown to light yellowish
brown, coxa with some black maculae. Abdomen yellow, with dark margins (Fig. 7G).
Ootheca. Light reddish brown. Surface with longitudinal lines. Serrations of the
keel small and bluntly rounded. Respiratory canals absent (Fig. 7A, B).

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
82
Natural history. is new species can be found in dry soil under clis along the
roadside (Fig. 7C, D).
Etymology. e species epithet is from similis (Latin) indicating this species is
similar to Eupolyphaga yunnanensis.
Figure 6. Eupolyphaga simila Qiu, sp. nov., male holotype A habitus, dorsal view B habitus, ventral view
C pronotum, dorsal view D head, ventral view E supra-anal plate, ventral view F subgenital plate (right
stylus missing), ventral view G genitalia, dorsal view H right phallomere, right-ventral view. Scale bars:
1.0 cm (A, B); 0.2 cm (C , D); 0.1 cm (E–H).

Species delimitation based on morphology and molecular analysis 83
Remarks. Due to the outbreak of COVID-19, all living females of the new spe-
cies were uncared-for in the lab during the research time. us, no female specimens
survived for further study, but LQ has taken a photograph of a female in the wild
(Fig.7H), which provides a chance to compare the coloration with the female of
Figure 7. Eupolyphaga simila Qiu, sp. nov. A ootheca, lateral view B serrations of ootheca C, D habitats
of Eupolyphaga simila, Siboguo Village, Lixian County, Sichuan Province E, F the living holotype after
emergence G the ventral side of a nymph H the ventral side of a female. Scale bars: 0.1 cm (A); 0.01 cm
(B). Photographs C–H by LQ.

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
84
E.yunnanensis. We also distinguished E. simila Qiu sp. nov. from E. yunnanensis by
using DNA barcoding; the genetic divergence of the two species being 13.86–14.82%,
and the ABGD analysis also supports the establishment of this new species.
Eupolyphaga sinensis (Walker, 1868)
Fig. 8
Polyphaga sinensis Walker, 1868: 14.
Homoeogamia sinensis Saussure, 1869: 282; Hollier et al. 2020: 347. Synonymized by
Qiu et al. (2018).
Heterogamia sinensis: Dohrn 1888: 132.
Heterogamia dohrniana Saussure, 1893: 309; Hollier et al. 2020: 345.
Polyphaga limbata Kirby, 1903: 379.
Eupolyphaga sinensis: Chopard 1929: 262; Qiu et al. 2018: 5 (revision); Qiu et al.
2019: 11 (checklist).
Type locality. “North China”.
New material examined. C · 2 males; Hubei Prov., Xiangyang City, Xianshan
Mountain; July 2020; Mao Ye leg; SWU-B-CC-010028 to 010029 · 1 male; Hubei
Prov., Wuhan City, Huangling District, Sushan Temple; 16 August 2019; Chen-Liang
Li leg.; SWU-B-CC-010030.
Distribution. China (Beijing, Hebei, Henan, Inner Mongolia, Liaoning, Jilin,
Tianjin, Shaanxi, Shanxi, Ningxia, Shandong, Jiangsu, Anhui, Hubei (new record),
Hunan, Chongqing, Guizhou, Yunnan).
Remarks. is species is newly recorded from Hubei Province. is is the most widely
distributed Eupolyphaga species in China, but in general it is more common in Northern
China than in the south. e distribution of this species in south China needs to be further
surveyed in the future. e ventral abdomen of this species in males is usually uniformly
yellowish white, while a specimen from Wuhan City has dark brown markings on the legs
and abdomen (Fig. 8B, F). By sequencing the COI genes of both individuals from Wuhan
and northern China (Beijing and Liaoning), we found that the genetic distances between
the individuals from Wuhan and north China reached 8.81% (Beijing) to 9.54% (Liaon-
ing); the ABGD analysis treats the Wuhan individual as a separated MOTU from individu-
als from northern China. Currently only one specimen is available from Wuhan. erefore,
we tentatively regard this variation as an intraspecic dierence.
Eupolyphaga hanae Qiu, Che & Wang, 2018
Eupolyphaga hanae Qiu, Che & Wang., 2018: 16; Qiu et al. 2019: 11 (catalogue).
Type locality. “Sichuan Prov., Chengdu City, Dujiangyan Prefectural-Level City, Da-
guan Town, Puzhao Temple; 770 m.”

Species delimitation based on morphology and molecular analysis 85
Figure 8. Eupolyphaga sinensis, male from Sushan Temple, Wuhan City, Hubei Province A habitus,
dorsal view B habitus, ventral view C a living male D head, ventral view E pronotum, dorsal view F legs
and abdomen (showing dark brown markings), ventral view G supra-anal plate, ventral view H subgenital
plate, ventral view I genitalia, dorsal view J right phallomere, right-ventral view. Scale bars: 1.0 cm (A, B);
0.2 cm (D–J). Photographs C by Chen-Liang Li.

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
86
New material examined. C · 2 males, 1 female, 10 nymphs, 4 oothecae;
Sichuan Prov., Luzhou City, Gulin County, Jianzhu Township, Daheidong Sce-
nic Area, Xiazhai; 899 m; 28°03.50'N, 105°34.70'E, 1 February 2019, Lu Qiu leg.;
SWU-B-CC-010031 to 010043 · 1 male, Sichuan Prov., Aba Prefecture, Wenchuan
County, Yingxiu Town; 1000 m; 17 September 1983; Xue-Zhong Zhang leg.; SWU-
B-CC-010044 · 2 males, Chongqing City, Jiangjin District, Simianshan Mountain,
Dawopu; 31 August 2018; Lu Qiu leg.; SWU-B-CC-010045 to 010046 · 1 male,
Chongqing City, Jiangjin District, Simianshan Mountain; 22 October 2006, Wei-Wei
Zhang leg.; SWU-B-CC-010047 · 1 male, Chongqing City, Jiangjin District, Simian-
shan Mountain; August 2018; De-Yao Zhou leg.; SWU-B-CC-010048.
Remarks. is species is mainly distributed in Sichuan, Chongqing. We obtained
COI sequences of this species from various localities (Simianshan Mountain and Jinyun-
shan Mountain in Chongqing, Shehong, Dujiangyan and Gulin in Sichuan). Genetic dis-
tances ranged from 2.02–6.12% between samples from these localities (Suppl.material 1).
Eupolyphaga robusta Qiu, Che & Wang, 2018
Figs 4A, C, 9
Eupolyphaga robusta Qiu, Che & Wang., 2018: 19; Qiu et al. 2019: 11 (catalogue).
Type locality. “Sichuan Prov., Aba Prefecture, Wenchuan County; 1100 m.”
New material examined. C · 2 males, 1 female, 1 nymph, 6 oothecae; Si-
chuan Prov., Aba Prefecture, Maoxian County, Nanxin Town, Miancu Village; 1518m;
31°35.73'N, 103°44.97'E; 7 August 2019; Zong-Qing Wang, Lu Qiu, Wei Han,
Huan-Yu Ren leg.; SWU-B-CC-010049 to 010052 · 1 nymph, 1 ootheca; Sichuan
Prov., Aba Prefecture, Maoxian County, Nanxin Town, Miancu Village; 1503 m;
31°35.77'N, 103°45.00'E; 2 October 2019; Lu Qiu, Hao Xu, Zhi-Teng Chen leg.;
SWU-B-CC-010053 · 1 male, 1 female, 10 oothecae; Sichuan Prov., Aba Prefecture,
Maoxian County, Xiaomiao Mountain; 1650 m; 31°40.85'N, 103°51.33'E; 6 August
2019; Lu Qiu, Wei Han, Huan-Yu Ren leg.; SWU-B-CC-010054 to 010055 · 1 male,
1 female, 1 nymph, 4 oothecae; Sichuan Prov., Aba Prefecture, 013 Township road
near Wenchuan County; 1473 m; 31°28.63'N, 103°35.90'E; Zong-Qing Wang, Lu
Qiu, Wei Han, Huan-Yu Ren leg.; SWU-B-CC-010056 to 010057 · 2 males, 6 fe-
males, 2 oothecae; Sichuan Prov., Aba Prefecture, Wenchuan County, Miansi Town;
29 March 2020; Jian-Yue Qiu leg.; SWU-B-CC-010058 to 010065.
Supplementary description. Male: Tegmina almost black, with a few white macu-
lae, some individuals have more and larger maculae that are randomly distributed on
tegmina. Abdomen orange or black, if black, the terminal two segments dimly yellowish.
Female: Body length 29.1–32.5 mm, body width 20.5–22.5 mm. Body uniformly
black (Fig. 9C, D). Antennal sockets, ocelli, ante-clypeus white. e base and end of labrum
white, the middle black. Ocelli small, nearly triangular. Distance between ocelli smaller
than distance between antennal sockets, interocular space wide. Pullivi and arolia absent.
Hind margin of supra-anal plate protruded, medially with a large incision (Fig. 9C, D).

Species delimitation based on morphology and molecular analysis 87
Nymph: Similar to female, blackish brown.
Ootheca: Yellowish brown. Surface with shallow longitudinal lines. Serrations on
the keel large and curved, compactly arranged. Respiratory canals indistinct (Fig. 9I, J).
Natural history. is species is distributed in the Minjiang River basin of Sichuan,
which is a typical arid river valley. It can be found in multiple habitats, such as in soil,
under abandoned bricks or rocks, or inside dead wood.
Remarks. is species was originally described based on a single male specimen
collected in 1983. e holotype is dark brown, the last two segments of abdomen are
yellowish white, and its vertex has a small yellowish spot. During two expeditions in
Minjiang River basin in 2019, we obtained numerous specimens of this species. We
found that the fresh samples of the males are black rather than dark brown. e macu-
lae on tegmina show some variation, most male individuals are black, with sparse whit-
ish maculae, but there are individuals with larger maculae. We also found that most of
Figure 9. Eupolyphaga robusta from Minjiang River basin A male, dorsal view B male, ventral view
Cfemale, dorsal view D female, ventral view E–G habitats of Eupolyphaga robusta, Maoxian County,
Sichuan Province H the variation of the abdominal coloration in males I ootheca, lateral view J serrations
of ootheca. Scale bars: 1.0 cm (A–D); 0.1 cm (I); 0.01 cm (J). Photographs E–H by LQ.

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
88
the newly collected individuals dier from the holotype by the coloration of abdomen.
Most males have a uniformly orange abdomen, while the specimens from Miansi Town
have the same abdominal color as the holotype, i.e., the apical two segments of abdo-
men are yellowish, the remaining portions are black. Among the newly collected male
specimens, no individual was found with a yellow spot at vertex, which indicates the
yellow spot at vertex in the holotype may be a variation or aberration.
We sequenced the COI marker from eight samples and found that the genetic
distances between samples from Miansi Town (male with black abdomen) and sam-
ples from other localities (males with orange abdomens) are somewhat distant (3.29–
3.57%), while the genetic distances between samples with orange abdomen are much
lower (0.15–0.47%) (Suppl. material 1).
Eupolyphaga hupingensis Qiu, Che & Wang, 2018
Figs 10A–D, 11
Eupolyphaga hupingensis Qiu, Che & Wang., 2018: 18; Qiu et al. 2019: 11 (catalogue).
Type locality. “China: Hunan: Daling Village, Hupingshan Town, Shimen County”,
444 m.
New material examined. C · 1 male, 2 females, 1 ootheca; Hunan Prov.,
Zhangjiajie City, Sangzhi County, Badagongshan Nature Reserve, Tianpingshan
Mountain; 1400 m; 29°46.99'N, 110°05.44'E; 30–31 July 2019; Hao Xu, Jian-Yue
Qiu leg.; SWU-B-CC-010066 to 010068 · 1 male; Anhui Prov., Jinzhai County, Ma-
zongling; 580 m; 2 August 2018, Yu-Chen Zheng leg.; SWU-B-CC-010069.
Supplementary description. Female: Body length 32.3–41.0 mm, body width
21.5–23.7 mm. Body uniformly dark reddish brown. Antennal sockets, ocelli, ante-
clypeus, and labrum white. Ocelli large. Distance between ocelli almost equal to in-
terocular space, and slightly narrower than distance between antennal sockets. Arolia
and pulvilli absent. Posterior margin of supra-anal plate protruded, medially with a
large incision (Fig. 10A, B).
Nymph. Similar to female, blackish brown.
Ootheca. Reddish brown. Longitudinal lines distinct and evenly arranged
(Fig.10C). Serrations of keel large, strongly curved, the space between the nearest two
serrations compact, each serration with weak respiratory canals (Fig. 10D).
Distribution. China: Hunan, Anhui (new record).
Remarks. e individual from Jinzhai County (North Anhui) is almost morpho-
logically identical to other specimens of E. hupingensis, only the color of its clypeus and
labrum, abdomen and legs are lighter than those of holotype (Fig. 11B, C), and styli
are slender (Fig. 11F). However, the COI genetic distance between individuals from
Jinzhai and Shimen County (North Hunan) reached 8.21%. Because only one speci-
men is available from Jinzhai, we temporarily regard it as intraspecic variation and
look forward to obtaining more samples for further investigation.

Species delimitation based on morphology and molecular analysis 89
Figure 10. Eupolyphaga hupingensis from Mt. Badagongshan, Hunan Province (A–D) and Eupolyphaga
dongi from Mt. Gaoligongshan, Baoshan City, Yunnan Province (E–H). A female, dorsal view B female, ven-
tral view C ootheca, lateral view D serrations of ootheca E female, dorsal view F female, ventral view Goo-
theca, lateral view H serrations of ootheca. Scale bars: 1.0 cm (A, B , E , F); 0.2 cm (C, G); 0.05 cm (D, H ).

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
90
Eupolyphaga dongi Qiu, Che & Wang, 2018
Fig. 10E–H
Eupolyphaga dongi Qiu, Che & Wang, 2018: 52; Qiu et al. 2019: 11 (checklist).
Type locality. “Yunnan Prov., Baoshan City, Longyang District, Nankang Village;
1980 m.”
New material examined. C · 1 male; Yunnan Prov., Baoshan City, Mt. Gao-
ligongshan Nature Reserve, Baihualing; 13 April 2017; Zhi-Wei Dong leg.; SWU-
Figure 11. Eupolyphaga hupingensis, male from Mazongling, Jinzhai County, Anhui Province A habitus,
dorsal view B habitus, ventral view C head, ventral view D tegmen, ventral view E supra-anal plate, ven-
tral view F subgenital plate, ventral view G genitalia, dorsal view H right phallomere, right-ventral view.
Scale bars: 1.0 cm (A, B, D ); 0.2 cm (C , G , H); 0.1 cm (E, F).

Species delimitation based on morphology and molecular analysis 91
B-CC-010070 · 2 females, 1 nymph, 8 oothecae; Yunnan Prov., Baoshan City, Mt.
Gaoligongshan Nature Reserve, Baihualing; 20–21 June 2020; Lu Qiu, Jin-Lin Liu
leg.; SWU-B-CC-010071 to 010073.
Supplemental description. Female: Body length 20.1–21.2 mm, body width
14.6–14.9 mm. Body dark brown, with small fuzzy markings (Fig. 10E). Face with a
large black spot, vertex and antennal sockets forming a pale-colored frame enclosing
the large black spot. Ocelli degraded and not distinct. Tibial spines reddish brown,
pulvilli present, arolia absent. Abdomen with some inconspicuous pale-colored mark-
ings and a pale-colored midline. e posterior edge of the supra-anal plate protruded,
median emargination small. Posterior margin of subgenital plate wide, with an incon-
spicuous central shallow concavity (Fig. 10E, F).
Nymphs: Similar to female.
Ootheca: Reddish brown. Surface with obvious longitudinal lines (Fig. 10G). Ser-
rations short and thick, nearly triangular, terminus blunt, without respiratory canals
(Fig. 10H).
Natural history. is species can be found in dry soil under huge rocks in the for-
est of Baihualing.
Discussion
In recent years, ABGD has been successfully applied in the species delimitation of some
Blattodea genera (e.g., Cryptocercus, Bai et al. 2018; Ectobiidae, Che et al. 2017; Margattea,
He et al. 2021; Anaplecta, Deng et al. 2020, Zhu et al. 2022). So far, species of Corydioidea
have rarely been studied by molecular species delimitation methods (Trotter et al. 2017). In
this study, the ABGD method was used in support of morphological species delimitation
of Eupolyphaga, and the results were consistent except for E. sinensis and E. hupingensis.
Samples of E. sinensis from three dierent localities (Beijing, Liaoning, and Wuhan)
were divided into two MOTUs. Compared to the genetic distance between individuals
from Beijing and Liaoning (4.76%), the individual from Wuhan was signicantly more
genetically distant from the rst two (8.81% and 9.54%, respectively) (Suppl. material
1). Although the color of the abdomen and legs of the Wuhan individual (Fig. 8A–C)
diers from those of other E. sinensis, its other characteristics (including male genitalia)
exactly coincide with those characteristics of E. sinensis. For E. hupingensis, which was
also divided into two MOTUs by the ABGD method, the genetic distance between
Anhui and Hunan samples reached 8.21%. However, the dense maculae on the tegmina,
the brown-black but slightly yellow abdomen on the last two segments (Fig.11A, B, D)
and the robust L3 (Fig. 11G) suggest that these two MOTUs belong to the same species.
Alternatively, the delimitation results of ABGD of these two species (E. sinensis and E.
hupingensis) could suggest that there may be cryptic species. Since only one male sample
from Wuhan and Jinzhai were collected, we temporarily regard them as intraspecic
variation and reserve for obtaining more samples for more in-depth research.
Not all species have as unusually large intraspecic genetic distances as E. sinensis
and E. hupingensis. For E. hanae, E. yunnanensis, and E. robusta, samples from dierent

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
92
geographic populations were not signicantly dierent morphologically, and their genetic
distances (E. hanae, 2.02%–6.12%; E. yunnanensis, 0.30%–2.01%; E. robusta, 0.15%–
3.57%, Suppl. material 1) were much lower than that with E. sinensis and E.hupingensis.
e genus Eupolyphaga is widely distributed in China, especially in the mountainous
regions of Western China. It is still under-researched due to its secretive habits, which make
it not easily detected in the natural environment. e low dispersal ability and the habit of
inhabiting only in specic micro-habitats may lead to their long-term geographical isola-
tion (Qiu et al. 2018), and their homogeneous habitat (dry soil and humus) is hardly af-
fected by external environment changes. ese two facts may lead to genotypic divergence
over phenotypic divergence, and ultimately cause relatively large genetic distances between
species without much change in external morphology. We speculate there are still a large
number of undiscovered species, and a more extensive eld survey of the vast unexplored
mountainous areas in Western China is still needed. In the future, studies of this genus can
be more comprehensive by incorporating female external genitalia and other characters.
Acknowledgements
We sincerely thank Yu-Chen Zheng (China Agricultural University), Wei-Wei Zhang,
Dong Wang (Chongqing), Bing-Qiang Wang (Beijing), De-Yao Zhou (Shanghai), Hao
Xu, Jian-Yue Qiu (Mianyang Normal University), Zhi-Teng Chen (Jiangsu University
of Science and Technology), Mao Ye (Xiangyang, Hubei), Chen-Liang Li (Wuhan,
Hubei), Lu-Yu Wang, Jin-Lin Liu, Huan-Yu Ren (SWU), Qi Li (Chengdu, Sichuan),
and Lei Wang (Suining, Sichuan) for their eorts in specimen collecting. We thank Dr.
John Richard Schrock for proofreading the draft. We also cordially thank Dr. Frédéric
Legendre, Dr. Tobias Kovacs, and an anonymous reviewer for their numerous com-
ments and revisionary suggestions. is study is supported by the National Natural
Sciences Foundation of China (Nos. 32070468, 31872271).
References
Bai QK, Wang LL, Wang ZQ, Lo N, Che YL (2018) Exploring the diversity of Asian
Cryptocercus (Blattodea: Cryptocercidae): species delimitation based on chromosome
numbers, morphology and molecular analysis. Invertebrate Systematics 32(1): 69–91.
https://doi.org/10.1071/IS17003
Che YL, Gui SH, Lo N, Ritchie A, Wang ZQ (2017) Species delimitation and phylogenetic
relationships in ectobiid cockroaches (Dictyoptera, Blattodea) from China. PLoS ONE
12(1): e0169006. https://doi.org/10.1371/journal.pone.0169006
Chopard L (1929) Orthoptera palaearctica critica VII. Les Polyphagiens de la faune paléarc-
tique (Orth., Blatt.). Eos 5: 223–358.
Deng WB, Liu YC, Wang ZQ, Che YL (2020) Eight new species of the genus Anaplecta
Burmeister, 1838 (Blattodea: Blattoidea: Anaplectidae) from China based on molecu-

Species delimitation based on morphology and molecular analysis 93
lar and morphological data. European Journal of Taxonomy 720: 77–106. https://doi.
org/10.5852/ejt.2020.720.1117
Dohrn H (1888) Neue und wenig bekannte Blattiden. Stettin Entomological Society 49: 129–
132.
Feng PZ, Woo FC (1988) ree new species and two new records of Blattaria from Yunnan and
Guizhou, China. Entomotaxonomia 10: 305–312.
Folmer O, Black M, Wr H, Lutz R, Vrijenhoek R (1994) DNA primers for amplication
of mitochondrial cytochrome c oxidase subunit i from diverse metazoan invertebrates.
Molecular Marine Biology and Biotechnology 3(5): 294–299.
He JJ, Jin DT, Wang YS, Che YL, Wang ZQ (2021) Species delimitation of Margattea cock-
roaches from China, with seven new species (Blattodea, Ectobiidae, Pseudophyllodromii-
nae). ZooKeys 1036: 121–151. https://doi.org/10.3897/zookeys.1036.63232
Hollier J, Marshall J, Legendre F (2020) An annotated list of the Blattodea (Insecta) described
by Henri de Saussure. Part 1: e Corydiidae. Revue Suisse de Zoologie 127(2): 341–348.
https://doi.org/10.35929/RSZ.0023
Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: Multiple sequence align-
ment, interactive sequence choice and visualization. Briengs in Bioinformatics 4(20):
1160–1166. https://doi.org/10.1093/bib/bbx108
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions
through comparative studies of nucleotide sequences. Journal of Molecular Evolution
16(2): 111–120. https://doi.org/10.1007/BF01731581
Kirby WF (1903) XXXVII.-Notes on Blattidæ &c., with Descriptions of new Genera and Spe-
cies in the Collection of the British Museum, South Kensington.-No. II. Annals & Maga-
zine of Natural History 7(70): 373–381. https://doi.org/10.1080/00222930308678873
Klass KD (1997) e external male genitalia and the phylogeny of Blattaria and Mantodea.
Bonner Zoologische Monographien 42: 1–341.
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analy-
sis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870–1874.
https://doi.org/10.1093/molbev/msw054
Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2017) PartitionFinder 2: New
methods for selecting partitioned models of evolution for molecular and morphologi-
cal phylogenetic analyses. Molecular Biology and Evolution 34: 772–773. https://doi.
org/10.1093/molbev/msw260
Li XR, Zheng YH, Wang CC, Wang ZQ (2018) Old method not old-fashioned: Parallel-
ism between wing venation and wing-pad tracheation of cockroaches and a revision of
terminology. Zoomorphology 137(4): 519–533. https://doi.org/10.1007/s00435-018-
0419-6
Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and eective
stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology
and Evolution 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
Puillandre N, Lambert A, Brouillet S, Achaz G (2012) ABGD, Automatic Barcode Gap Dis-
covery for primary species delimitation. Molecular Ecology 21(8): 1864–1877. https://doi.
org/10.1111/j.1365-294X.2011.05239.x

Wei Han et al. / ZooKeys 1120: 67–94 (2022)
94
Qiu L, Che YL, Wang ZQ (2018) A taxonomic study of Eupolyphaga Chopard, 1929 (Blattodea:
Corydiidae: Corydiinae). Zootaxa 4506(1): 1–68. https://doi.org/10.11646/zootaxa.4506.1.1
Qiu L, Yang ZB, Wang ZQ, Che YL (2019) Notes on some corydiid species from China, with the
description of a new genus (Blattodea: Corydioidea: Corydiidae). Annales de la Société En-
tomologique de France 55(3): 261–273. https://doi.org/10.1080/00379271.2019.1603081
Roth LM (2003) Systematics and phylogeny of cockroaches (Dictyoptera: Blattaria). Oriental
Insects 37(1): 1–186. https://doi.org/10.1080/00305316.2003.10417344
Saussure HD (1869) Mélanges Orthoptérologiques. Memoires de la Société de physique et
d’histoire naturelle de Genève 20: 227–328.
Saussure HD (1893) Revision de la tribu des Hétérogamiens (Orthoptéres de la Famille des Blat-
tides). Revue Suisse de Zoologie 1(2): 289–318. https://doi.org/10.5962/bhl.part.3746
Trotter AJ, McRae JM, Main DC, Finston TL (2017) Speciation in fractured rock landforms: towards
understanding the diversity of subterranean cockroaches (Dictyoptera: Nocticolidae: Nocticola)
in Western Australia. Zootaxa 4250(2): 143–170. https://doi.org/10.11646/zootaxa.4250.2.2
Walker F (1868) Catalogue of the Specimens of Blattariæ in the Collection of the British Mu-
seum. British Museum, London, 239 pp. https://doi.org/10.5962/bhl.title.8495
Wang YS, Chen R, Jin DT, Che Y-L, Wang Z-Q (2021) New record of Cyrtonotula Uvarov,
1939 (Blaberidae, Epilamprinae) from China, with three new species based on morphologi-
cal and COI data. ZooKeys 1021: 127–143. https://doi.org/10.3897/zookeys.1021.59526
Woo FC (1987) Investigations on domiciliary cockroaches from China. Acta Entomologica
Sinica 4: 430–438.
Woo FC, Feng PZ (1992) Blattoptera: Corydiidae, Blattidae, Epilampridae and Phyllodromii-
dae. In: Chinese Academy of Since (Org.) Insects of the Hengduan Mountains Region, 1.
Science Press, Beijing, 865 pp.
Yang R, Wang ZZ, Zhou YS, Wang ZQ, Che YL (2019) Establishment of six new Rhabdoblatta
species (Blattodea, Blaberidae, Epilamprinae) from China. ZooKeys 851: 27–69. https://
doi.org/10.3897/zookeys.851.31403
Zhu J, Zhang JW, Luo XX, Wang ZQ, Che YL (2022) ree cryptic Anaplecta (Blattodea,
Blattoidea, Anaplectidae) species revealed by female genitalia, plus seven new species from
China. ZooKeys 1080: 53–97. https://doi.org/10.3897/zookeys.1080.74286
Supplementary material 1
Table S1
Author: Wei Han, Lu Qiu, Jing Zhu, Zong-Qing Wang, Yan-Li Che
Data type: Genetic distance.
Explanation note: Genetic distances between COI gene of 45 Eupolyphaga species.
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e 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.1120.87483.suppl1