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ZooKeys 1166: 315-331 (2023)
DOI: 10.3897/zookeys.1166.103055

Research Article

A new species of Achalinus Peters, 1869 (Squamata, Xenodermidae)
from Hunan Province, China

Shun Ma’*®, Sheng-Chao Shi'”*®, Sun-Jun Xiang2®, Fu Shu*®, Jian-Ping Jiang!2©

er wo NY

Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
University of Chinese Academy of Sciences, Beijing 100049, China

College of Biological and Food Engineering, Huaihua University, Huaihua 418000, China
Central South Academy of Inventory and Planning of NFGA, Changsha 410014, China

Corresponding author: Jian-Ping Jiang (jiangjp@cib.ac.cn)

OPEN Qrceess

Academic editor: Robert Jadin
Received: 6 March 2023
Accepted: 2 May 2023
Published: 13 June 2023

ZooBank: https://zoobank.
org/11887A71-8708-46AB-AB6D-
B864779D5075

Citation: Ma S, Shi S-C, Xiang S-J,
Shu F, Jiang J-P (2023) A new
species of Achalinus Peters, 1869
(Squamata, Xenodermidae) from

Hunan Province, China. ZooKeys 1166:

315-331. https://doi.org/10.3897/
zookeys.1166.103055

Copyright: © Shun Ma 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).

Abstract

A new species, Achalinus hunanensis sp. nov., is described from middle and western
Hunan Province based on the results of molecular systematics and morphological char-
acters. It diverges from known congeners by a significant genetic divergence (p-dis-
tance 3.2%-16.9% based on CO1 mitochondrial gene), and it can be distinguished from
all known congeners by the following morphological characters: (1) all dorsal scales
strongly keeled, 23 rows throughout the body, the outmost one strongly keeled and en-
larged; (2) tail relatively short, TaL/TL 0.221 ~ 0.225; (3) maxillary teeth 23; (4) the suture
between internasals 2 x as long as that between prefrontals; (5) loreal one, subrectan-
gular, LorH/LorL 0.62 ~ 0.70; (6) supralabials 6, the 4 and 5‘ touch the eye; (7) the two
anterior temporals in contact with eye; (8) ventrals 163-165, subcaudals 69-72, not
paired. This raises the number of known species of Achalinus to 24.

Key words: Achalinus hunanensis sp. nov., morphology, phylogeny, divergence time,
taxonomy

Introduction

The odd-scaled snake genus Achalinus Peters, 1869, which is widely distributed
in northern Vietnam, China, and Japan, is a group of small to medium-sized, noc-
turnal, fossorial, and non-venomous snakes (Zhao et al. 1998; Zhao 2006). Itis the
most diverse genus of six known genera of the family Xenodermidae: 23 species
in Achalinus; two species in Fimbrios Smith, 1921; two species in Parafimbrios
Teynié, David, Lottier, Le, Vidal & Nguyen, 2015; one species in Paraxenodermus
Deepak, Lalronunga, Lalhmingliani, Das, Narayanan, Das & Gower, 2021; two spe-
cies in Stoliczkia Jerdon, 1870; one species in Xenodermus Reinhardt, 1836 (Ha
et al. 2022; Yang et al. 2022; Zhang et al. 2023). Among Achalinus species, more
than half (14/23) have been described since 2019 (Wang et al. 2019; Ziegler et
al. 2019; Li et al. 2020; Luu et al. 2020; Miller et al. 2020; Hou et al. 2021; Huang
et al. 2021; Li et al. 2021; Ha et al. 2022; Yang et al. 2022; Zhang et al. 2023).
Besides, previous studies revealed that A. ater Bourret, 1937, A. formosanus

* These authors contributed equally to this work.

315

Shun Ma et al.: A new Achalinus species

Boulenger, 1908, A. huangjietangi Huang, Peng & Huang, 2021, A. niger Maki,
1931, A. rufescens Boulenger, 1888, and A. spinalis Peters, 1869 required further
studied, due to their distinct morphology variation, deep intraspecific divergence,
or non-monophyletic relationship (Zhao et al. 1998; Miller et al. 2020; Huang et
al. 2021; Zhang et al. 2023). However, because of poor sampling, morphological
and molecular information of Achalinus were lacking, leading to unresolved taxo-
nomic problems and poorly known distribution patterns. Therefore, the diversity
of this genus is underestimated and further research is required.

During our field work, two specimens were collected from Hunan Province,
China (Fig. 1). They could be assigned to Achalinus by body small, slender, and
cylindrical; whole body scales strongly keeled, lanceolate-shaped, and metallic
luster; and preocular and postocular absent, loreal and temporals contacting
the eyes directly. However, further morphological and molecular analyses re-
vealed that these specimens comprise a separate taxon from other known spe-
cies. Thus, we described them as a new species herein.

Materials and methods
Molecular phylogenetic analyses

Two specimens of the genus Achalinus were collected in Hunan Province, Chi-
na: CIB 119039 was collected in Huaihua City, and CIB 119040 was collected
in Ningxiang County, and they were deposited in Chengdu Institute of Biology
(CIB) of Chinese Academy of Sciences (CAS). Genomic DNA were extracted from
preserved muscle tissues of them using QlAamp DNA Mini Kit (QIAGEN, Chang-
sheng Biotechnology Co. Ltd). A fragment of the mitochondrial cytochrome c
oxidase subunit 1 (CO1) was amplified using the primer pairs dglco and dghco
(Meyer et al. 2005). The polymerase chain reaction (PCR) was performed in 25
UL of reactant with the following cycling conditions: 95 °C for 4 min; 35 cycles of
denaturing at 95 °C for 30 s, annealing at 48 °C for 30 s, and extending at 72 °C
for 60 s; and a final extending step of 72 °C for 10 min (Wang et al. 2019). PCR
products were sequenced by Beijing Qingke New Industry Biotechnology Co., Ltd.

For phylogenetic analysis, 25 sequences (Table 1) were selected, among
which 23 sequences (NO. 3-25) were obtained from National Center for Bio-
technology Information (NCBI) including 22 sequences from 19 species of
Achalinus and three sequences of Fimbrios klossi Smith, 1921, Parafimbrios
lao Teynié, David, Lottier, Le, Vidal & Nguyen, 2015 and Xenodermus javanicus
Reinhardt, 1836, which were used as outgroups (Luu et al. 2020; Ha et al. 2022).

CO1 sequences (681 bp) were input in MEGA11 (Tamura et al. 2021) and
aligned by MUSCLE (Edgar 2004), and then the uncorrected pairwise distances
(p-distance) were calculated in MEGA11. IQ-TREE 1.6.12 was used to conduct the
maximum likelihood (ML) analysis (Nguyen et al. 2015) under the best-fit model
TN+F+l+G4 selected by Modelfinder according to BIC (Kalyaanamoorthy et al.
2017). Ultrafast Bootstrap Approximation (UFB) node support as assessed by us-
ing 5000 ultrafast bootstrap replicates and the UFB = 95 was considered signifi-
cantly supported (Hoang et al. 2018). The single branch tests were made using
SH-like approximate likelihood ratio test (SH-aLRT) via 1000 replicates, and the
SH = 80 was also considered supported well (Stephane et al. 2010). For Bayesian
inference (Bl), the best-fitting model HKY+I+G was selected by jModelTest 2.1.10

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 316

Shun Ma et al.: A new Achalinus species

110°0'0" 112°0'0" 114°0'0"

BE ek yale A* hunanensis Sp. nov. 2

he Fons Type locality :

ue @ A. hunanensis sp. nov.
> ningshanensis
Type locality
: Elevation ;
5 High : 3636 S
=) i
= oD
= [or 2 386

S b
= o
Q o
” o
S S
- Oo
©O ©0
a NS
S &
= S
a ©
si NS

110°0'0" 112°0'0" 114°0'0"
Figure 1. Distribution of Achalinus hunanensis sp. nov. and its sister taxon A. ningshanensis. Blue pentacle: the type
locality of A. ningshanensis: Xunyangba, Ningshan County, Shaanxi Province, China. Red triangle: the type locality of
A. hunanensis sp. nov. (CIB 119039): Hecheng District, Huaihua City, Hunan Province, China. Red circle: A. hunanensis sp.
nov. (CIB 119040): Wazizhai, Ningxiang County, Changsha City, Hunan Province, China.

identified via BIC (Darriba et al. 2012) on CIPRES (Miller et al. 2010). The Bayesian
inference analysis was conducted using MrBayes v. 3.2.1 (Ronquist et al. 2012)
under HKY+I+G this model, and four chains run was calculated for 10 million gen-
erations, sampled every 1000 with the first 25% of samples discarded as burn-in,
resulting in a potential scale reduction factor (PSRF) of < 0.005. Bayesian poste-
rior probabilities (BI) = 0.95 were considered supported well.

We also estimated divergence time by BEAST v. 2.6.7 using CO1 sequenc-
es dataset (Bouckaert et al. 2019). Three calibration constraints were used:
the divergence time between Achalinus and other Xenodermatidae: 38.6 Mya;
the divergence time between Xenodermus and Fimbrios + Parafimbrios: 29.67

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 ang

Shun Ma et al.: A new Achalinus species

Table 1. Localities, voucher information, GenBank numbers, and references for all samples used in this study.

NO. Species Locality Voucher CO1 GenBank No. References

1 A. hunanensis sp. nov. Huaihua, Hunan, China CIB 119039 0Q848425 This study

2 A. hunanensis sp. nov. Ningxiang, Hunan, China CIB 119040 0Q848426 This study

3 A. ningshanensis Ningshan, Shaanxi, China ANU 20220006 ON548422 Yang et al. 2022
4 A. ningshanensis Ningshan, Shaanxi, China ANU 20220007 ON548423 Yang et al. 2022
5 A. ater Huaping Nature Reserve, Guangxi, China SYS r00852 MN380334 Wang et al. 2019
6 A. dehuaensis Dehua, Fujian, China YBU 13013 MZ442662 Li et al. 2021

7 A. emilyae Hoanh Bo, Quang Ninh, Vietnam IEBR 4465 MK330857 Ziegler et al. 2019
8 A. formosanus Taiwan, China RN2002 KU529452 Unpublished

9 A. huangjietangi Huangshan, Anhui, China HSR18030 MT380191 Huang et al. 2021
10 | A. juliani Ha Lang, Cao Bang, Vietnam IEBR A.2018.8 MK330854 Ziegler et al. 2019
11 A. meiguensis Mianyang, Sichuan, China GP835 MZ442641 Li et al. 2021
12 | A. niger Taiwan, China RN0667 KU529433 Unpublished
13. | A. panzhihuaensis Yanbian, Sichuan, China KIZ 040189 MW664862 Hou et al. 2021
14. | A. pingbianensis Honghe, Yunnan, China YBU 18273 MT365521 Li et al. 2020
15 | A. rufescens Hongkong, China SYS r001866 MN380339 Wang et al. 2019
16 | A. spinalis Badagong Mountains, Hunan, China SYS 1001327 MN380340 Wang et al. 2019
17. = A. timi Thuan Chau, Son La, Vietnam IEBR A.2018.10 MK330856 Ziegler et al. 2019
18 | A. tranganensis Ninh Binh, Vietnam VNUF R.2018.21 MW023086 Luu et al. 2020
19 | A. vanhoensis Van Ho, Son La, Vietnam VNUF R.2019.13 ON677935 Ha et al. 2022
20 | A. yangdatongi Wenshan Nature Reserve, Yunnan, China KIZ 034327 MW664865 Hou et al. 2021
21 A. yunkaiensis Dawuling Forestry Station, Guangdong, China SYS r001443 MN380329 Wang et al. 2019
22 | A.zugorum Bac Me, Ha Giang, Vietnam IEBR 4698 MT502775 Miller et al. 2020
23 ‘| Fimbrios klossi Quang Ngai, Vietnam IEBR 3275 KP410744 Teynié et al. 2015
24 | Parafimbrios lao Louangphabang, Laos MNHN 2013.1002 KP410746 Teynié et al. 2015
25 | Xenodermus javanicus Sumatera Barat, Indonesia: = KP410747 Teynié et al. 2015

Mya; and the divergence time between Fimbrios and Parafimbrios: 17.66 Mya
(Kumar et al. 2022). Two independent searches of 20 million generations were
conducted, sampling every 1000 iterations with 25% of the initial samples dis-
carded as burn-in. Tracer v. 1.7.2 was used to evaluate estimate ESS for all
parameters (Rambaut et al. 2018).

Morphological examination

Morphological data of known species of Achalinus were obtained from the two
newly collected specimens, examination of museum specimens (A. ater: n = 1;
A. rufescens: n = 1; A. yunkaiensis: n = 1) (Appendix 1) and many key references
(Boulenger 1888, 1893, 1896; Denburgh 1912; Bourret 1935, 1937; Hu and Zhao
1966; Hu et al. 1973; Koshikawa 1982; Zong and Ma 1983; Ota and Toyama
1989; Zhao et al. 1998; Zhao 2006; Wang et al. 2019; Ziegler et al. 2019; Li et al.
2020; Luu et al. 2020; Miller et al. 2020; Yu et al. 2020; Hou et al. 2021; Huang
et al. 2021; Li et al. 2021; Chen et al. 2022; Ha et al. 2022; Xu et al. 2023; Yang
et al. 2022; Zhang et al. 2023).

Morphological descriptions followed Zhao (2006) and Yang et al. (2022).
Three characters were measured to the nearest 1 mm by Deli Stainless Ruler (No.

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 318

Shun Ma et al.: A new Achalinus species

8460): snout-vent length (SVL), tail length (TaL) and total length (TL); the other
characters were measured to the nearest 0.01 mm by Deli Digital Vernier Caliper
(DL91150): head length (HL), head width (HW), eye horizontal diameter (ED), loreal
height (LorH), loreal length (LorL), length of the suture between internasals (LSBI),
length of the suture between prefrontals (LSBP). We counted the following ratios:
TaL/TL: tail length/total length, LorH/LorL: loreal height/loreal length, LSBI/LSBP:
length of the suture between internasals/length of the suture between prefrontals,
HL/HW: ratio head length/head width. We also directly compared the length of the
sutures between internasals and prefrontals (LSBI vs. LSBP).

Scalation features and their abbreviations are as follows: loreals (Loreal),
supralabials (SPL), infralabials (IFL), the number of chin shield pairs (Chins),
the number of infralabials touch the first pair of chin shields (IFL-1% Chin), su-
praoculars (SPO), temporals (TEM), the number of anterior temporals touch
the eye (aTEM-Eye) (those head bilateral scale counts were given as left/right),
pre-ventral scales (PrV), ventral scales (VEN), subcaudal (SC), entire or divided
of the anal (Anal), dorsal scale rows (DSR) (counted at one-head-length behind
the head, at midbody, at one-head-length before the anal). We also counted the
number of maxillary teeth (MT) under the microscope.

Results
Molecular systematics

All Achalinus samples cluster in a monophyletic group with high supporting
values (SH 100/ UFB 100/ BI 1), and they can be divided into six clades, al-
though the relationships among these clades are still unresolved (Fig. 2). Clade
A contains A. dehuaensis Li, Wu, Xu. Zhu, Ren, Guo & Dong, 2021; clade B con-
tains A. emilyae Ziegler, Nguyen, Pham, Nguyen, Pham, Van Schingen, Nguyen
& Le, 2019, A. rufescens and A. tranganensis Luu, Ziegler, Ha, Lo, Hoang, Ngo,
Le, Tran & Nguyen, 2020; clade C A. zugorum Miller, Davis, Luong, Do, Pham,
Ziegler, Lee, De Queiroz, Reynolds & Nguyen, 2020, A. huangjietangi, A. panzhi-
huaensis Hou, Wang, Guo, Chen, Yuan & Che, 2021, A. meiguensis Hu & Zhao,
1966, A. spinalis, A yunkaiensis Wang, Li & Wang, 2019, A. niger and A. formosa-
nus; Clade D includes A. timi Ziegler, Nguyen, Pham, Nguyen, Pham, Van Schin-
gen, Nguyen & Le, 2019 and A. vanhoensis Van Ha, Ziegler, Sy, Le, Nguyen & Luu,
2022, and Clade E contains A. pingbianensis Li, Yu, Wu, Liao, Tang, Liu & Guo,
2020, respectively. Clade F consists five species with a significantly high nodal
support (SH 100/ UFB 100/ BI 1; divergence time: 1.76 Mya, 95% highest pos-
terior density interval (HPD): 2.473 ~ 0.988 Mya), of which, the two specimens
newly collected in this work are firstly clustered together as a lineage with well
support (SH 88/UFB 96/BI 0.98; divergence time: 0.21 Mya, 95% HPD: 0.534
~ 0.003 Mya), and then clustered with A. ningshanensis Yang, Huang, Jiang,
Burbrink, Gong, Yu, Zhang, Huang & Huang, 2022 (SH 98/UFB 100/BI 1; diver-
gence time: 0.48 Mya, 95% HPD: 0.914 ~ 0.120 Mya), forming a sister group of
A. yangdatongi Hou, Wang, Guo, Chen, Yuan & Che, 2021 diverged at 0.83 Mya
(95% HPD: 1.418 ~ 0.279 Mya), and then they form a sister group of the lineage
which contains the A. ater and A. juliani Ziegler, Nguyen, Pham, Nguyen, Pham,
Van Schingen, Nguyen & Le, 2019 (divergence time: 1.27 Mya, 95% HPD: 1.930
~ 0.602 Mya) (Fig. 3).

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 319

Shun Ma et al.: A new Achalinus species

F 100/100/1

KIZ 034327 A. yangdatongi
SYS r00852 A. ater
85/88/0.93 IEBR A.2018.8 A juliani
YBU 18273 A. pingbianensis
D 100/100/1 rae A.2018.10 A. timi
VNUF R.2019,13 A. vanhoensis

100/100/1 RN2002 A. formosanus

Naess A, niger
SYS r001443 A. punkaiensis
SYS r001327 A. spinalis
84/-10.52 99/99/1 | GP835 A. meiguensis
KIZ, 040189 A. panzhihuaensis
SR18030 A. Auangjietangi
IEBR 4698 A. zugerum
IEBR 4465 A. emilyae
SYS r001866 A. rufescens
VNUF R.2018.21 A. franganensis
YBU 13013 A. dehuaensis

100/100/1 —— IEBR 3275 Fimbrios klosst

76/76/0.62

82/58/0.53

79/-/0.78 | 66/74/-

89/83/0.95

0.06 100/100/1

-/82/0.70

A

100/100/1

Xenodermus javanicus MNHN 2013.1002
Parafimbrios lao

Figure 2. Phylogenetic tree of the genus Achalinus inferred from CO1 gene fragment (681 bp) using Maximum Likelihood.
The tree nodes present the supporting values: SH-like approximate likelihood ratio test, Ultrafast Bootstrap Approxima-
tion and Bayesian posterior probabilities, respectively (SH, %/UFB, %/BI) (the ones lower than 50 are displayed as “-”).
Achalinus ningshanensis is noted in blue and A. hunanensis sp. nov. is noted in red.

0.21
(0.534, 0.003]

0.48
10.914, 0.120]

0.15
[0.382, 0.004]
1.418, 0.279]

Clade F

1.27
1.930, 0.602]

KIZ 034327 A. yangdatongi

0.77
1.428, 0,152|
IEBR A. 2018.8 A. juliani

1.76 "

|2.473, 0.988]

SYS r00852 A, ater
2.04

[2.807, 1.203]

83) Clade D+E
2.29
[3.099, 1.524]
a a TT | Clade B
38.56 Clade A+C
139.139, 37.987] 070)
tf IEBR 3275 Fimbrios klossi
17.64
[18.202, 17.023]
90:68 th MNHN 2013.1002 Parafimbrios laos

[30.236, 29.059]

th Ken odermnts fVANICUS
L

| |
40 30 15 5 0 Mya

Figure 3. Divergence date estimation (Mya) and 95% HPD (in square bracket) of Clade F. Achalinus ningshanensis is in
blue and A. hunanensis sp. nov. is in red.

Zookeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 320

Shun Ma et al.: A new Achalinus species

The genetic distances range from 5.0% (A. timi and A. vanhoensis) to 18.1%
(A. dehuaensis and A. meiguensis) among the known Achalinus species studied
in this work (Table 2), of which, within the Clade F, the genetic distances among
the four known species range from 5.8% (A. ningshanensis and A. yangdatongi) to
9.6% (A. juliani and A. ningshanensis), while the lineage composed by those two
newly collected specimens range from 3.2% to 8.8% divergent from congeners.

The results above indicate that the Hunan samples are close to the species
A. ningshanensis but consist independent evolution lineage.

Morphological systematics

The two specimens of the genus Achalinus newly collected from Hunan Province
can be easily distinguished from all other known congeners (Tables 3, 4). By hav-
ing 23-23-23 dorsal scale rows, the two specimens can be distinguished from
these species including A. formosanus formosanus Boulenger, 1908 (vs. 29-27-
25), A. f. chigirai Ota & Toyama, 1989 (vs. (25-27)-(25-27)-25), A. meiguensis
(vs. (21-23)-(19-21)-(19-21)), A. niger (vs. 25-25-23), A. panzhihuaensis (vs.
23-23-19), A. timi (vs. 25-25-23), A. tranganensis (vs. 25-23-23), A. vanhoensis
(vs. 25-23-23), A. zugorum (vs. 25-23-23). By having loreal separated from pre-
frontal, they are different from A. jinggangensis and A. pingbianensis (vs. loreal
fused with prefrontal). By having LSBI vs. LSBP > 1, they differ from A. dabie-
shanensis Zhang, Liu, Huang, Hu, Yu, Sun, Zhang, Wen & Zhang, 2023 (vs. < 1),

Table 2. Uncorrected p-distances (%) among Achalinus species inferred from mitochondrial CO1 gene.

1-2 A. hunanensis
Sp. nov.

3-4 A.
ningshanensis

5A. ater

6 A. dehuaensis
7A. emilyae

8 A. formosanus
9 A. huangjietangi
10 A. juliani

11 A. meiguensis
12 A. niger

13 A.
panzhihuaensis

14 A. pingbianensis
15 A. rufescens

16 A. spinalis

17 A. timi

18 A. tranganensis
19 A. vanhoensis
20 A. yangdatongi
21 A. yunkaiensis

22 A. Zugorum

1-2
0.5

3.2-3:3

7.1-7.3
15.1-15.2
12.9-13.3
13.8-13.9
16.8-16.9
8.7-8.8
16.4
t3:2=13.3
16.2

11.1-11.2
12.1-12.2
14.0-14.3
12.1
13.7-14.2
11.3-11.7
5.1
11.7-12.1
11.6-11.9

3-4

0.7

7.6-7.7
16.3-16.5
13.5-14.1
14.8-15.1
17.2
9.1-9.6
17.0
14.6
17.1-17.4

11.7-12.4
12.3-12.7
15.1-15.6
13.6
14.3-15.2
12.1-12.4
eo
$3-0513-7
12.8

5

16.5
11.7
14.1
15.0
7.1

15.4
13.5
16.2

11.8
12.7
15.2
13.3
12.7
13.1
6.2

12.8
13.1

6

15.7
15.7
16.8
15.2
18.1
15.7
15.3

14.8
14.3
14.3
15.8
14.2
15.8
14.0
14.7
14.3

7

14.6
14.8
11.5
15.4
12.9
16.6

13.0
8.0
13.9
12.9
10.6
12.3
12.8
13.1
12.9

8

16.2
13.4
15.6
9.0

16.0

14.5
14.1
13.9
14.0
17.3
14.1
14.4
12.3
13.7

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055

9

14.8
15.2
14.6
18:2

13.0
14.3
13.4
14.8
13:7
14.8
14.6
12.5
14.4

10

16.8
12.9
Ta

12.2
12.3
BS
13.7
12.3
13.5
ces)

12.5
13

11

13.9
11.6

16.8
17.3
16.0
15.8
16.4
15.6
17.1
15.8
15.0

12

14.4

11.7
12.7
13.5
12.0
14.9
12.6
13.7
12.2
13.4

13

14.9
16.0
15.8
N A5)e5)
16.4
15.5
15.5
15:7
NSH

14

12.9
13:3
12.2
13.3
10.8
11.3
11.6
10.9

15

13.0
13:9
11.5
13.8
11.5
13.3
13:8

16

14.3
14.6
12.9
14.2
12.0
13.3

17

13.5
5.0

13.1
14.1
13.4

18 | 19 | 20 | 21

13:3

12.8 | 11.3

13.5 | 13.6 | 12.0
12.5 | 12.0 | 12.2 | 10.9

22

321

Shun Ma et al.: A new Achalinus species

Table 3. Main morphological characters of Achalinus hunanensis sp. nov.

Joueherhunse CIB 119039 CIB 119040
Holotype Paratype
Sex Male | Male
SVL 255 204
TaL 74 | 58
TL 329 262
TaL/TL 0.225 | 0.221
Loreal 1/1 1/1
LorH 1.03/1.04 0.91/0.93
LorL 1.54/1.58 1.46/1.49
LorH/LorL 0.70/0.66 0.62/0.62
LSBI 1.78 1.52
LSBP 0.88 0.76
LSBI/LSBP 2.02 | 2.00
LSBI vs. LSBP >1 >1
HL 7.91 6.33
HW 4.80 3.38
HL/HW 1.65 1.87
ED 1.42/1.41 1.37/1.36
MT 23 23
SPL 3-2-1/3-2-1 3-2-1/3-2-1
IFL 5/6 5/5
IFL-1% Chin 3/4 3/3
SPO 1/1 1/1
TEM 2+244/24+2+4 24+244/24+2+4
aTEM-Eye 2/2 2/2
PrV 2 Ds
VEN 163 165
Sc 69 72
Anal Entire Entire
DSR 23-23-23 23-23-23

A. hainanus Huang, 1975 (vs. = 1), A. huangjietangi (vs. < 1), A. spinalis (vs. < 1),
A. werneri Van Denburgh, 1912 (vs. = 1) and A. yunkaiensis (vs. = 1). By having
more ventrals (163-165), they can be distinguished from A. dehuaensis (vs.
142-154), A. emilyae (157-161), and A. rufescens (vs. 132-156).

Within the clade F, the two newly collected specimens from Hunan can be
identified from A. ater by having nostril in the anterior part of the nasal (vs. nostril
in the posterior part of the nasal), loreal length ~ 1.5 x than loreal height (vs. lore-
al length > 2 x than loreal height), and more subcaudals (69-72 vs. 47-70). They
are different from A. juliani by having different dorsal scale rows (23-23-23 vs. 25-
23-23), less maxillary teeth (23 vs. 28), and less subcaudals (69-72 vs. 77-91).
They differ from A. yangdatongi by having relatively shorter tail length in males
(0.221 ~ 0.225 vs. 0.261 ~ 0.262), more ventrals in males (163-165 vs. 155), and
fewer subcaudals in males (69-72 vs. 76) fewer maxillary teeth (23 vs. 24-26).
They also can be easily distinguished from its sister group A. ningshanensis by
the following morphological characters: (1) the suture between the internasals
2 x as long as the suture between the prefrontals vs. the suture between the

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 322

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Shun Ma et al.: A new Achalinus species

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055

Shun Ma et al.: A new Achalinus species

internasals subequal to the suture between the prefrontals; (2) relatively longer
tail (TaL/TL: 0.221 ~ 0.225 vs. 0.121 ~ 0.161); (3) more subcaudals: 69-72 vs.
41-46; (4) two chin pairs vs. three chin pairs; (5) relatively narrow and long lore-
als (0.62 ~ 0.70 vs. 0.45 ~ 0.58) (more details are presented in Table 5).

Combined the results of molecular systematics and morphological charac-
ters above, the specimens newly collected in this work represent a new spe-
cies, and we describe it herein.

Table 5. Main morphological characters of Achalinus hunanensis sp. nov. and A. ning-
shanensis.

Species Achalinus hunanensis sp. nov. A. ningshanensis
Sex Males (n = 2) Females (n = 5)
SVL 204-255 334-463
TaL 58-74 62-72
TL 262-329 398-527
TaL/TL 0.221 ~ 0.225 0.121 ~ 0.161
Loreal 1/1 1/1
LorH/LorL 0.62 ~ 0.70 0.45 ~ 0.58
LSBI/LSBP 2.00 ~ 2.02 0.95 ~ 1.11
LSBI vs. LSBP >1 =1
HL 6.33-7.91 11.17-13.72
HW 3.38-4.80 4.75-7.48
HL/HW 1.65-1.87 1.78-2.67
MT 23 >
SPL 3-2-1 3-2-1
IFL 5-6 5
Chins 2 3
IFL-1* Chin 3-4 2=3
SPO 1 1
TEM 24+2+4 24+243/242+4/2+3+4
aTEM-Eye 2 1-2
VEN 163-165 159-174
Sc 69-72 41-46
Anal Entire Entire
DSR 23-23-23 23-23-23 (rarely 21)
References This study Yang et al. 2022

Taxonomic account

Achalinus hunanensis sp. nov.
https://zoobank.org/05012A7E-84CE-4C6D-A915-51FCF365DFFE
Figs 4,5

Chresonymy. Achalinus ater: Shu et al. 2014; Shen et al. 2014; Gao et al. 2022.

Material examined. Holotype. CIB 119039 (Collection No. 20130505001),
subadult male (Fig. 4), collected in early May 2013, by Sheng-Chao Shi and
Sun-Jun Xiang from Huangyan Village, Hecheng District, Huaihua City, Hunan
Province, China (27°28'N, 110°02'E; ca. 880 maz.s.l.).

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 324

Shun Ma et al.: A new Achalinus species

A

Smm Smm

Figure 4. The holotype (CIB 119039, subadult male) of Achalinus hunanensis sp. nov. A dorsolateral view B ventral view
C right side of middle body view D dorsal head view E left side of head view F right side of head view G ventral head view.
Photographs by SCS.

Paratype. CIB 119040, subadult male (Fig. 5), collected on 16 June 2022, by
Sheng-Qiang Liu from Wazizhai, Ningxiang County, Changsha City, Hunan Prov-
ince, China (28°00'N, 111°53'E; ca. 1020 m a.s.I.).

Etymology. This new species is named after its known distribution range,
which is endemic to Hunan Province. The Chinese name is suggested as
“Sips Are” (HU Nan Ji Shé) and the English name “Hunan Odd-scale Snake” or
“Hunan Burrowing Snake” is suggested.

Diagnosis. (1) 23 rows of dorsal scales throughout the body, all dorsal scales
strongly keeled, and the outmost one strongly keeled and enlarged; (2) tail rela-
tively short, TaL/TL 0.221 ~ 0.225; (3) maxillary teeth 23; (4) the suture between
internasals 2 x as long as that between prefrontals; (5) loreal one, subrectan-
gular, LorH/LorL 0.62 ~ 0.70; (6) supralabials six, the 4° and 5 touch the eye;
(7) the two anterior temporals in contact with eye; (8) ventrals 163-165, sub-
caudals 69-72, not paired.

Description of holotype. A subadult male with a total length of 329 mm
(SVL 255 mm and TaL 74 mm); tail relatively short, Tal/TL 0.225; body slender,

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 325

Shun Ma et al.: A new Achalinus species

A C

10mm 5mm

Figure 5. The paratype (CIB 119040, subadult male) of the Achalinus hunanensis sp. nov. A dorsolateral view B ventral
view C dorsal head view D right side of head view E ventral head view. Photographs by SCS.

cylindrical; head length (HL) 7.91 mm, head width 4.80 mm, HL/HW 1.65, slight-
ly distinct from neck; eye small, ED 1.42/1.41 mm; maxillary teeth 23, small,
equally sized and curved. Rostral small, triangular, only the upper tip visible
from above. Length of the suture between the internasals (LSBI 1.78 mm) ~ 2 x
as long as length of the suture between the prefrontals (LSBP 0.88 mm). Nostril
in the anterior part of the nasal. Loreal one, subrectangular, loreal height (LorH)
1.03/1.04 mm, loreal length (LorL) 0.70/0.66 mm, LorH/LorL 0.62 ~ 0.70. Su-
praocular one. Frontal one, pentagonal, pointed backwards, much shorter than
the parietals. Parietals paired and elongated. No preoculars and postoculars.
Temporals 2+2+4, the anterior two contact the eye, the lower anterior temporal
much larger, the upper medium temporal much larger, the upper posterior tem-
poral much larger and separated from the other side one by two small scales
which contact the parietals. Supralabials 6, 4° and 5" contact the eye, the last
one much elongated. One mental. Two chin shields, similar length. Infralabials
5/6, the first one contact with each other after the mental and before the 1°
chin shields, 1s'-3'¢/1st—4" touch the 1: chin shields.

Dorsal scales lanceolate and strongly keeled; 23 rows throughout the body;
those of the outmost rows on both sides significantly enlarged and strongly
keeled. Ventrals 163, with two preventrals; anal entire; subcaudals 69, not paired.

Coloration of holotype. In life, dorsum dark, slightly metallic, vent black-
brown, dark brown near the margin, grey in the margin. A yellowish brown patch

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 326

Shun Ma et al.: A new Achalinus species

on the head occipital. The head ventral anterior part dark brown and posterior
part yellowish white (Shu et al. 2014). In preservation, dorsum brown, vent ante-
rior part grey and posterior part light brown. Ventral side of tail brown. The head
ventral anterior part brown and posterior part grey.

Variations. Main morphological characters were listed in Table 3. The oth-
er sample are very similar to the holotype except that: (1) more ventrals: CIB
119040: 165; (2) more subcaudals: CIB 119040: 72; (3) vent coloration: CIB
119040: dark brown throughout the vent.

Distribution and habits. Achalinus hunanensis sp. nov. is currently only
known from Hunan Province, China: Hecheng District, Huaihua City and Ningx-
iang County, Changsha City (880-1020 m a.s.I.). The holotype was found at
night, near a mountain stream (AT 24 °C, RH 80%) with shrubs under subtropical
evergreen broadleaves forest. It was moving from leaf litter to the stream.
Earthworms were found at the same place, which we speculated as its prey
(Shu et al. 2014).

Discussion

Based on molecular evidence, the newly collected Achalinus specimens in
this study are most closely to A. ningshanensis but a genetic differentiation
(p-distance 3.2%) already exists between these two groups (Fig. 2, Table 2).
The newly collected Achalinus specimens and its sister group A. ningshan-
ensis have separate distribution ranges at south of Yangtze River and north
of Yangtze River, respectively, isolated by Three Gorges (Fig. 1). In addition,
their estimated divergence time was at 0.48 Mya (Fig. 3), which broadly co-
incides with the formation of Three Gorges of the Yangtze River (0.30 ~ 0.12
Mya) (Zhang et al. 2018), hindering their communication and driving allopat-
ric speciation. Moreover, combining their distinct morphological differences
(Table 5), we describe them as a new species. Currently, 24 Achalinus spe-
cies are reported.

Achalinus ater was first recorded in Hunan Province only based one spec-
imen (Shen et al. 2014; Shu et al. 2014; Gao et al. 2022). However, this study
found that this record was a misidentification and this specimen was designed
as the new species holotype. Therefore, A. ater recorded on Hunan reptile check
list should be transferred as A. hunanensis.

Due to the secretive life history and morphological similarities, many cryptic
species may be “hidden” within known widely distributed species, such as A.
spinalis, A. rufescens, and A. ater (Wang et al. 2019; Yang et al. 2022), and the
description of A. hunanensis sp. nov. indicated that further study is necessary
to conduct by using different Achalinus species and geographic populations to
revise the mystery snakes and reveal their evolutionary history.

Acknowledgements

The study was supported by the National Key Programme of Research and De-
velopment, Ministry of Science and Technology (2022YFF1301401). We thank
the support of CIB Herpetological Museum, and sincerely express our thanks
to Mr. Sheng-Qiang Liu for his kind and careful work in the field survey. We also
thank Qi-Heng Chen for his help in molecular phylogenetic analyses.

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 327

Shun Ma et al.: A new Achalinus species

Additional information

Conflict of interest

No conflict of interest was declared.

Ethical statement

No ethical statement was reported.

Funding

National Key Programme of Research and Development, Ministry of Science and Tech-
nology (2022YFF1301401).

Author contributions

Shun Ma: Laboratory work, methodology, data analysis, validation, writing: orgination
and draft, writing: review and editing; Sheng-Chao Shi: methodology, investigation and
resources, writing: orgination and draft, writing: review and editing; Sun-Jun Xiang: in-
vestigation and resources, writing: review and editing; Fu Shu: investigation and resourc-
es, writing: review and editing; Jian-Ping Jiang: conceptualization, data curation, project
administrition, resources, supervision, writing: review and editing.

Author ORCIDs

Shun Ma @ https://orcid.org/0009-0003-861 1-4550
Sheng-Chao Shi © https://orcid.org/0000-0003-2337-6572
Sun-Jun Xiang © https://orcid.org/0009-0003-6692-7087
Fu Shu © https://orcid.org/0000-0002-6082-8112
Jian-Ping Jiang © https://orcid.org/0000-0002-1051-7797

Data availability

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

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Appendix 1

Examined Achalinus specimens

A. ater (n = 1): China, Guizhou Province, Xingyi County: CIB 631115243.
A. rufescens (n = 1): China, Hong Kong: CIB 119042.
A. yunkaiensis (n = 1): China, Hunan Province, Xinning County: CIB 119041.

ZooKeys 1166: 315-331 (2023), DOI: 10.3897/zookeys.1166.103055 331