ZooKeys 1210: 173–195 (2024)

DOI: 10.3897/zookeys.1210.128121

Research Article

Two new species of freshwater goby (Teleostei, Gobiidae) from

the Upper Youshui River, Chongqing, China
Lingzhen Li1,2* , Chaoyang Li1,2*, Weihan Shao3, Suxing Fu1,2 , Chaowei Zhou1,2

1 College of Fisheries, Southwest University, Chongqing, China

2 Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development
(Ministry of Education), Southwest University, Chongqing 400715, China
3 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, China
Corresponding author: Chaowei Zhou (zcwlzq666@163.com)

Abstract

Two previously unknown species of Rhinogobius have been discovered in the streams
of the Upper Youshui River, within the Yuan River Basin, Xiushan County, Chongqing,
China. These new species are named as Rhinogobius sudoccidentalis and Rhinogobius
lithopolychroma. Phylogenetic analysis based on mitochondrial genomes revealed that
R. sudoccidentalis is genetically closest to R. reticulatus, while R. lithopolychroma shares
the greatest genetic similarity with R. leavelli. Morphological distinctions allow for the
clear differentiation of these species. Rhinogobius sudoccidentalis sp. nov. is character-
ized by having VI–VII rays in the first dorsal fin and I, 8–9 rays in the second dorsal fin. The
longitudinal scale series typically consists of 22–24 scales, while the transverse scale
series comprises 7–8 scales. Notably, the predorsal scale series is absent and the total
vertebrae count is 12+17=29. Rhinogobius lithopolychroma sp. nov. can be distinguished
from other species by the presence of 13–15 rays on the pectoral fin. Its longitudinal scale
series ranges from 30 to 33 scales, with no scales in the predorsal area. The total verte-
bral count is 30, with 12 precaudal and 18 caudal vertebrae. The head and body of this
Academic editor: Tihomir Stefanov
species are light gray with irregular orange markings on the cheeks and opercle. Through
Received: 22 May 2024
morphological and molecular analyses, it has been confirmed that R. lithopolychroma and
Accepted: 9 July 2024
Published: 22 August 2024 R. sudoccidentalis represent novel species within the Rhinogobius genus.

ZooBank: https://zoobank. Key words: China, fish taxonomy, Gobiidae, Gobionellinae, mitochondrial genome, Yuan-
org/43C7344A-120B-4BE5-A7CB- jiang River Basin
0107B18DDB9D

Citation: Li L, Li C, Shao W, Fu S,
Zhou C (2024) Two new species of
freshwater goby (Teleostei, Gobiidae) Introduction
from the Upper Youshui River,
Chongqing, China. ZooKeys 1210: The genus Rhinogobius, belonging to the subfamily Gobionellinae within the
173–195. https://doi.org/10.3897/ family Gobiidae, is widely distributed across East and Southeast Asia. First de-
zookeys.1210.128121 scribed by Gill in 1859, with Rhinogobius similis Gill, 1859 as the type species, this
genus is known for its high species richness. Over 92 valid species have been
Copyright: © Lingzhen Li et al. described, with an increasing number of new species being discovered. In recent
This is an open access article distributed under
terms of the Creative Commons Attribution
years, several new species of Rhinogobius have been found in China, including
License (Attribution 4.0 International – CC BY 4.0). R. houheensis Kunyuan et al., 2020, R. coccinella Endruweit, 2018, R. maculagenys

* These authors contributed equally to this work.

173
Lingzhen Li et al.: Two new species of freshwater goby

Wu et al., 2018, R. maxillivirgatus Xia et al., 2018, R. nanophyllum Endruweit, 2018,

R. wuyanlingensis Huang et al., 2016, R. niger Huang et al., 2016, R. immaculatus
Li et al., 2018, R. lintongyanensis Chen et al., 2022 and R. lianchengensis Wang
& Chen, 2022. To date, a total of 47 species of Rhinogobius have been recorded
in China (Chen et al. 2022a) . The significant diversity of Rhinogobius species in
China suggests that the overall species diversity within this genus may be un-
derestimated. Notably, the recent discoveries of Rhinogobius species have been
concentrated in East China, with fewer new species found in other regions.
The Upper Yuanjiang River Basin benefits from a favorable climate and encom-
passes numerous stream habitats within its mountainous areas. The biodiversity
in Xuan’en and Fanjingshan, traversed by the Upper Yuanjiang River Basin, is ex-
ceptionally high and potentially serves as a glacial refuge (Fei et al. 2017). Conse-
quently, it is inferred that the biodiversity in other regions of the Upper Yuanjiang
River Basin, particularly within its stream habitats, may have been underestimated.
During surveys conducted between June 2023 and January 2024 in the
streams of the Upper Youshui River within the Yuanjiang River Basin in Chongq-
ing, two species of Rhinogobius were discovered. Historically, only R. similis
and Rhinogobius cliffordpopei (Nichols, 1925) were documented in the Yuan-
jiang River Basin in Chongqing, with these species primarily adapted to lake
and reservoir environments (Wu et al. 2008; Suzuki et al. 2016). In contrast, the
newly discovered species exclusively inhabit streams and are characterized by
large eggs, unlike R. similis and R. cliffordpopei, which produce small eggs (Li
2011). The Upper Youshui River features a diverse stream ecosystem where
species distribution is influenced by factors such as substrate composition,
temperature, and current velocity. This study delves into the habitat of Rhinogo-
bius in the Upper Youshui River to explore the habitat segregation of Rhinogo-
bius, building upon previous research concerning the ecological preferences of
Rhinogobius species (Sone et al. 2001; Ito et al. 2006).

Materials and methods

Samples

A total of 44 specimens were collected from Chongqing Municipality and Guizhou

Province (Fig. 1) using a hand net. All specimens were preserved in 75% ethanol
and are stored at Southwest University in Rongchang District, Chongqing, China.

Morphometrics and meristic methods

Morphological measurements were primarily based on a previous study (Wu

et al. 2008). Data were collected from the left side of each fish using vernier
calipers, measuring 27 traits to the nearest 0.1 mm. Measurements included
the first dorsal fin, second dorsal fin, pectoral fin, anal fin, longitudinal scales,
transverse scales, and predorsal scales. Abbreviations for the cephalic sensory
pore system followed Chen and Kottelat (2005). The pattern of interdigitation
of the dorsal-fin pterygiophores and neural spines (P-V) was observed from
radiographs. The P-V method and vertebral counting were expressed using a
specific formula to describe the goby’s interdigitation pattern of dorsal-fin pte-
rygiophores and neural spines (Akihito et al. 1984). For example, in the formula

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 1. Map of the distribution of Rhinogobius sudoccidentalis sp. nov. and Rhinogobius lithopolychroma sp. nov. in Upper
Youshui River, with locations in southwest China shown in the lower right corner. Maps were prepared using ArcMap 10.8.

(P-V) 3/II II I I I 0/9: (P-V) stands for dorsal-fin pterygiophores and neural spines;
“3” indicates that three neural spines are anterior to the first pterygiophore; “II II
I I” indicates there are 2 pterygiophores between the neural spine of the 3rd and
4th vertebrae; 2 between the neural spine of the 4th and 5th vertebrae; 1 between
the neural spine of the 5th and 6th vertebrae; and 1 between the neural spine
of the 5th and 6th vertebrae; “0” indicates no pterygiophore between the neural
spines of the 7th and 8th vertebra; “9” indicates that the first pterygiophore of the
1st ray of the 2nd dorsal fin is inserted above the 9th vertebral body. Color in life
was described based on samples and photographs taken in fish tanks.

DNA sequencing and phylogenetic analysis

Four specimens were used for DNA barcoding. Total DNA was extracted from
the caudal fin following Maeda et al. (2021a) and Wanghe et al. (2020). Briefly,
single-stranded circular DNA molecules were amplified into a DNB (DNA Nano-
ball) containing more than 300 copies via rolling circle replication. These DNBs
were then applied to mesh pores on the chip using high-density DNA nano-chip
technology. Sequencing was performed by cPAS. Identification of complete
mitochondrial genomes from assembled contigs was achieved through two
criteria: 1) comparison with the complete mitochondrial genome of Stiphodon
alcedo Maeda, Mukai, & Tachihara, 2011 (accession: AB613000.1) (BLASTN
e-value ≤ 1e-100), and 2) confirmation that 100 bp of both the head and tail DNA
sequences of a contig were identical, indicating that the sequence was circular.

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Lingzhen Li et al.: Two new species of freshwater goby

Complete mitochondrial genomes were aligned using MAFFT v.7.244 (Katoh

and Standley 2013). The obtained mitochondrial gene was compared with Rhi-
nogobius wuyanlingensis Yang, Wu & Chen, 2008 (accession: NC_062781.1),
confirming identical sequences at the head and tail DNA regions, indicative of
circularity. Aligned mitochondrial genomes underwent phylogenetic analysis us-
ing maximum likelihood (ML) methods with RAxML v. 8.2.3 (Stamatakis 2014),
incorporating mitochondrial gene data from the GenBank library (Table 1).

Table 1. List of accession numbers and sequence length of mitochondrial genome sequences in this study.

Accession number Length of sequence (bp) Remarks

Rhinogobius estrellae LC648292 16682 Maeda et al. (2021b)
Rhinogobius estrellae LC648294 16504 Maeda et al. (2021b)
Rhinogobius estrellae LC648295 16505 Maeda et al. (2021b)
Rhinogobius estrellae LC648296 16504 Maeda et al. (2021b)
Rhinogobius tandikan LC648297 16691 Maeda et al. (2021b)
Rhinogobius tandikan LC648298 16690 Maeda et al. (2021b)
Rhinogobius tandikan LC648299 16918 Maeda et al. (2021b)
Rhinogobius tandikan LC648300 16690 Maeda et al. (2021b)
Rhinogobius similis LC648303 16499 Maeda et al. (2021b)
Rhinogobius similis LC648304 16499 Maeda et al. (2021b)
Rhinogobius formosanus MT363639 16500 Yang et al. (2020)
Rhinogobius formosanus MN549279 16502 Genbank
Rhinogobius szechuanensis OM617727 16492 Liu WZ et al. (2023)
Rhinogobius leavelli MH729000 16499 Zhang and Shen (2019)
Rhinogobius davidi OM617724 16627 Song et al. (2023)
Rhinogobius rubromaculatus KU674802 16503 Genbank
Rhinogobius flumineus LC648305 16504 Maeda et al. (2021b)
Rhinogobius flumineus LC648306 16503 Maeda et al. (2021b)
Rhinogobius yaima LC648307 16500 Maeda et al. (2021b)
Rhinogobius yaima LC648308 16500 Maeda et al. (2021b)
Rhinogobius yonezawai LC648309 16500 Maeda et al. (2021b)
Rhinogobius yonezawai LC648310 16500 Maeda et al. (2021b)
Rhinogobius nagoyae LC648315 16498 Maeda et al. (2021b)
Rhinogobius sp. MO LC648314 16499 Maeda et al. (2021b)
Rhinogobius brunneus LC648311 16500 Maeda et al. (2021b)
Rhinogobius brunneus LC648312 16500 Maeda et al. (2021b)
Rhinogobius wuyiensis OM678441 16502 Chen XJ et al. (2022b)
Rhinogobius lentiginis OM617725 16633 Chen XJ et al. (2022b)
Rhinogobius niger OM791349 16496 Genbank
Rhinogobius maculagenys OK545540 16500 Hu J et al. (2023)
Rhinogobius shennongensis OM961050 16500 Genbank
Rhinogobius cliffordpopei KX898434 16511 Genbank
Rhinogobius cliffordpopei KP694000 16529 Genbank
Rhinogobius cliffordpopei KT357638 16525 Genbank
Rhinogobius duospilus MH127918 16496 Tan et al. (2020)
Rhinogobius filamentosus OM678440 16510 Chen XJ et al. (2022b)
Rhinogobius wuyanlingensis OM617722 16491 Song et al. (2022)
Rhinogobius wuyanlingensis OM961051 16491 Genbank
Rhinogobius sp. Xiushan SRR28284919 16486 Collected in Xiushan, Chongqing
Rhinogobius lithopolychroma SRR28284920 16493 Collected in Xiushan, Chongqing
Rhinogobius sudoccidentalis SRR28284918 16480 Collected in Xiushan, Chongqing
Rhinogobius reticulatus SRR28284917 16497 Collected in Fuzhou, Fujian Province
Tridentiger kuroiwae LC653489 16501 Maeda et al. (2021b)
Tridentiger kuroiwae LC653490 16501 Maeda et al. (2021b)

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Lingzhen Li et al.: Two new species of freshwater goby

Outgroup specimens were analyzed using Tridentiger kuroiwae Jordan & Tana-
ka, 1927 (accessions: LC653489.1 and LC65349.1). The aligned mitochondrial
genomes from this study have been deposited in the GenBank library under
accession numbers SRR28284917-SRR28284920.

Results
Morphological analyses

Rhinogobius sudoccidentalis sp. nov.

https://zoobank.org/975F33AC-F810-4F32-8D57-26A583D924BB
Table 2, Figs 2–7

Type materials. Holotype. China • 1 ♂; Chongqing City, Xiushan County; 28°23'23"N,

108°53'16"E; 1 July. 2023; Lingzhen Li & Chaoyang Li leg.; RS20230001.
Paratypes. China - Chongqing City • 7 ♂♂, 3 ♀♀; Xiushan County; 28°23'23"N,
108°53'16"E; 1 July. 2023; Lingzhen Li & Chaoyang Li leg.; RS20230101 to 20230110.
• 4 ♂♂ ; Xiushan County; 28°26'17"N, 108°59'12"E; 1 July. 2023; Lingzhen Li & Ch-
aoyang Li leg.; RS20230111 to 20230114. • 1 ♂ , 1 ♀ ; Xiushan County; 28°24'51"N,
109°7'13"E ; 3 July. 2023; Lingzhen Li & Chaoyang Li leg.; RS20230115, 20230116.
• 1 ♂ , 2 ♀♀ ; Xiushan County; 28°22'30"N, 108°53'18"E; 4 July. 2023; Lingzhen Li &
Chaoyang Li leg.; RS20230118, 20230120. - Guizhou Province • 1 ♂ ; Tongren City;
28°8'50"N, 108°59'13"E; 3 July. 2023; Lingzhen Li & Chaoyang Li leg.; RS20230117.
Diagnosis. Rhinogobius sudoccidentalis can be distinguished from other
species in the genus by the following characteristics: it possesses VI–VII rays
in the first dorsal fin and I, 8–9 rays in the second dorsal fin. The longitudinal
scale series typically consists of 22–24 scales (most commonly 23), while the
transverse scale series typically comprises 7–8 scales (most commonly 8).
The predorsal scale series is absent. The total number of vertebrae counts is
12+17=29. Additionally, it features a black line stripe beneath the eye that ex-
tends to the mandible. Morphometrics Reference Table 2.
Description. Fins: The fins display distinct features: the first dorsal fin typ-
ically bears VI rays (18) or VII rays (2), while the second dorsal fin exhibits ei-
ther I, 8 rays (2) or I, 9 rays (18). The 3rd or 4th spine of the first dorsal fin is the
longest and lacks filamentous. In males, the depressed first dorsal fin extends
to the base of the 1st or 2nd branched ray of the second dorsal fin; in females,
it reaches only the base of the second dorsal fin anteriorly. The anal fin has I,
6 rays (1) or I, 7 rays (19), originating at a vertical line between the 2nd and 3rd
branched soft ray of the second dorsal fin. The pectoral fin typically has 14 rays
(2) or 15 rays (18) and is broad. In males, the rear tip of the pectoral fin aligns
parallel to the anus, a feature absent in females.
Scales: The body is covered with ctenoid scales, with enlarged mid-trunk
scales. The anterior predorsal area lacks scales, while the posterior occipital
region is adorned with cycloid scales. The belly is covered with small cycloid
scales. The longitudinal scale series ranges from 22 to 24 (mode: 23), and the
transverse scale series ranges from 7 to 8 (mode: 8). No scales are present in
the predorsal area.
Head canals: Pores σ are located between the anterior and posterior nares.
The anterior interorbital sections of oculoscapular canal are separated, fea-

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Lingzhen Li et al.: Two new species of freshwater goby

Table 2. Morphometrics of the types of R. sudoccidentalis expressed as a percentage

of standard length.

Variable Holotype Paratypes

Sex males males (N = 14) Females (N = 6)
Morphometry
Standard length (mm) 33.1 33.1–40.6(36.5) 30.2–36.5(32.1)
Head length (mm) 8.9 8.9–11.5(10.3) 7.3–9.9(8.1)
Percent standard length (%)
Head length 26.9 26.5–30.3(28.4) 23.7–27.1(25.2)
Predorsal length 37.8 31.7–43.1(37.4) 34.5–39.0(36.9)
Snout to second dorsal fin origin 53.8 53.6–59.2(56.2) 57.1–59.4(58.4)
Snout to anal fin origin 59.5 55.4–64.9(58.9) 59.3–64.7(62.7)
Snout to anus 54.1 51.2–56.9(53.5) 50.5–59.0(56.0)
Pre pelvic length 28.7 28.7–35.7(31.3) 28.8–33.7(30.6)
Caudal peduncle length 26.9 21.8–29.2(25.7) 17.3–27.5(23.2)
Caudal peduncle depth 8.8 8.2–10.5(9.2) 8.0–11.9(9.5)
First dorsal-fin base 8.5 8.5–17.3(12.8) 8.6–13.5(11.2)
Second dorsal-fin base 16.3 13.1–19.9(16.9) 14.6–19.6(16.1)
anal fin base 14.2 8.5–14.3(11.7) 8.7–11.7(10.0)
Caudal fin length 20.8 18.4–26.1(22.1) 13.5–23.8(18.5)
Pectoral fin length 20.2 19.6–24.1(21.8) 16.6–21.0(18.7)
Pelvic fin length 14.5 13.5–19.2(15.7) 12.5–18.1(15.8)
Body depth of pelvic fin origin 9.1 9.1–14.2(11.5) 9.8–12.9(11.7)
Body depth of anal fin origin 9.4 8.3–13.0(10.4) 9.3–11.5(10.6)
Pelvic fin origin to anus 26.9 22.0–27.2(25.2) 25.6–30.8(27.4)
Head depth 9.7 9.7–12.2(11.0) 9.6–12.9(11.0)
Percent head length (%)
Snout length 31.5 22.8–37.4(30.6) 19.2–31.6(25.5)
Eye diameter 14.6 11.3–19.3(14.2) 10.4–16.5(12.0)
Cheek depth 56.2 20.7–32.2(25.5) 21.2–29.1(24.1)
Postorbital length 55.1 43.1–60.4(51.7) 49.5–58.9(54.4)
Lower jaw length 31.5 27.9–48.7(38.7) 24.2–37.7(31.0)
Interorbital width 22.5 11.9–24.0(20.8) 12.1–19.5(16.3)
Head width in maximum 51.7 45.5–61.7(52.7) 50.5–65.8(58.0)

turing paired pore λ. A single pore κ is situated in the posterior region, with ω
present near posterior edge of eyes. There is an absence of ω1. The lateral
section of anterior oculoscapular canal exhibits pores α and terminal pore ρ.
The posterior oculoscapular canal ends with two terminal pores θ and τ. Pre-
opercular canals are presented, featuring pores ε, γ, and δ.
Sensory papillae: Row a extends anteriorly to just before the middle of the
eye. Row b is oblique and reaches forward to the posterior margin of the eyes.
Rows c and d are longer, extending behind the orbit, with Row cp positioned
between Rows c and d. Row f is paired. Opercular papillae include Rows ot, oi,
and os, with oi nearly reaching ot.
Vertebrae: The total vertebrae count is 12 + 17 = 29 (N = 5), with a (P–V)
pattern of 3/II II I I 0/9 (N = 5).

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 2. Dorsal (A), lateral (B), and ventral (C) views of preserved holotype of Rhinogobius sudoccidentalis sp. nov.
(RS20230001 male) and dorsal (D), lateral (E), and ventral (F) views of preserved paratype of Rhinogobius sudoccidenta-
lis sp. nov (RS20230101 female).

Figure 3. The skeletal system of R. sudoccidentalis sp. nov. Radiograph graphs of the whole body for paratype
RS20230102, male.

Coloration of preserved specimens: In males, the head and body of R. sudoc-

cidentalis exhibit a yellowish-brown color. There are paired brown stripes on
the snout converging at the tip, while the cheeks and opercle are adorned with
small black spots. A black stripe extends from under the eye to the mandible.
The ventral side displays dens coverage of small black spots. The membrane
of the first dorsal fin is gray, the second dorsal fin has a transparent membrane
with dense black mottling, and the anal fin exhibits a black membrane. The
pectoral fin is transparent. In females, the head and body are yellowish, with a
single black diagonal line below each eye. Irregular black patches are present
on the ventral side, and both the dorsal and anal fins are transparent.
Color in life: In males, the head and body of the R. sudoccidentalis are creamy
white. There are paired reddish-brown stripes on the snout meeting at the tip,
and the cheeks and opercle feature small black spots. A black stripe extends
from under the eye to the mandible. The ventral side is densely covered with
small orange spots. The membrane of the first dorsal fin is red with a blue mot-

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 4. Dorsal (A), lateral (B), and ventral (C) views of the head of the preserved holotype of R. sudoccidentalis sp. nov.
Red circles indicate sensory canal pores; red dots represent sensory papillae. Abbreviations: AN, anterior nare pore; PN,
posterior nare pore

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 5. Photographs of R. sudoccidentalis sp. nov. captured underwater in a tank A male B female. Photographed by Mr Zhi.

tling pattern between the 1st and 2nd spinous rays. The second dorsal fin has a
transparent membrane with dense black mottling and a white outer edge. The
anal fin exhibits a white margin with reddish dots on the ventral part of the red-
dish membrane. The pectoral fin is transparent, with a milky white basal portion.
In females, the head and body are yellowish, with paired brown stripes on the
snout meeting at the tip. There are single black diagonal lines below the eyes,
and irregular black patches on the ventral side. Both the dorsal and anal fin are
transparent, and the pectoral fin is transparent with a milky white basal portio.

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 6. Stream environment in Xiushan, Chongqing where R. sudoccidentalis sp. nov.

was collected.

Figure 7. Eggs of R. sudoccidentalis sp. nov. at the type locality.

Distribution and habitat. Rhinogobius sudoccidentalis was initially discov-

ered in a small stream in Xiushan, Chongqing, where it predominantly inhabits
areas characterized by large cobblestone substrates and slow-flowing water
at depths ranging from approximately 30 to 50 cm. Additionally, small popu-
lations of this species were also observed in Tongren, Guizhou Province. In
the Xiushan area, R. sudoccidentalis is the dominant fish species, utilizing the

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Lingzhen Li et al.: Two new species of freshwater goby

cobblestone bottom as an egg deposition site, with eggs characterized as large

(size 1.6–2.1 mm). During periods of high water levels in the creek, individuals
aggregate near the shore to seek refuge from the rapids.
Etymology. This species, discovered in Chongqing and Guizhou Province in the
southwestern region of China, has been named R. sudoccidentalis. The Latin roots
“sud” meaning “south” and “occidentalis” meaning “western” combine to signify
“southwestern”. The suggested Chinese name for this species is 西南吻虾虎鱼.

Rhinogobius lithopolychroma sp. nov.

https://zoobank.org/C1F210C4-1623-4B50-BB2A-F9DBAD7F197A
Table 3, Figs 8–13

Type materials. Holotype. China • 1 ♂; Chongqing City, Xiushan County; 28°21'21"N,

108°52'16"E; 2 July. 2023; Lingzhen Li & Chaoyang Li leg.; RL20230001.
Paratypes. China • Chongqing City • 6 ♂♂, 4 ♀♀; Xiushan County; 28°21'21"N,
108°52'16"E; 2 July. 2023; Lingzhen Li & Chaoyang Li leg.; RL20230101 to
20230110. • 11 ♂♂, 1 ♀; Xiushan County; 28°19'56"N, 108°52'17"E; 4 July. 2023;
Lingzhen Li & Chaoyang Li leg.; RL20230111 to 20230122.
Diagnosis. Rhinogobius lithopolychroma can be distinguished from other
species in the Rhinogobius by the following characteristics: It typically possess-
es 13–15 rays on the pectoral fin. The longitudinal scale series count ranges
from 30 to 33, with the predorsal area lacking scales. The total vertebrae count
is 30, comprising 12 precaudal and 18 caudal vertebrae. The head and body
of this species are light gray, adorned with irregular orange markings on the
cheeks and opercle. Morphometrics Reference Table 3.
Description. Fins: The fin configuration includes 6 rays on the first dorsal fin
(VI), with a 22 total rays. The second dorsal fin consists of one spine and either
9 or 10 branched rays, totaling 15 rays. The fourth or fifth spine of the first dorsal
fin is the longest and non-filamentous. In males, when the first dorsal fin is de-
pressed, the rear tip extends to the base of the second branched ray of the sec-
ond dorsal fin, while in females it reaches only to the base of the second dorsal
fin anteriorly. The anal fin has 1 spine and either 7 or 8 branched rays, totaling 13
rays. The origin of the anal fin is inserted at a vertical line between the first and
second branched soft ray of the second dorsal fin. The pectoral fins range from
13 to 15 rays, with 13 rays most common (present in 8 specimens), 14 rays in 13
specimens, and 15 in 1 specimen. The pectoral fins are broad in shape.
Scales: The body covered with ctenoid scales, with enlarged mid-trunk scales.
The anterior predorsal area lacks scales, while the posterior part of the occipi-
tal region is covered by cycloid scales. The belly is adorned with small cycloid
scales. The longitudinal scale series count ranges from 30 to 33, with a mode
of 31. The transverse scale series count ranges from 7 to 9, with a mode of 8.
Head canals: pores σ are located parallel to the anterior nares. The anterior
interorbital sections of the oculoscapular canal are separated, featuring paired
pore λ. There is a single pore κ in the posterior region, with ω present near pos-
terior edge of eyes and a lack of ω1. The lateral section of anterior oculoscap-
ular canal includes pores α and a terminal pore ρ. The posterior oculoscapular
canal possesses two terminal pores θ and τ. Preopercular canals are present-
ed, with pores ε, γ, and δ.

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Lingzhen Li et al.: Two new species of freshwater goby

Table 3. Morphometrics of the types of R. lithopolychroma expressed as a percentage

of standard length.

Variable Holotype Paratypes

Sex males males (N = 17) Females (N = 5)
Morphometry
Standard length (mm) 28.2 28.2–38.8(31.1) 27.5–36.4(33.6)
Head length (mm) 9.5 7.9–11.6(9.6) 7.9–10.7(9.7)
Percent standard length (%)
Head length 33.7 25.8–33.7(28.9) 25.8–30.3(28.8)
Predorsal length 36.5 28.2–43.4(37.2) 32.4–41.6(37.1)
Snout to second dorsal fin origin 58.2 42.0–58.5(54.6) 54.2–62.0(58.9)
Snout to anal fin origin 66.7 56.7–66.7(61.8) 63.5–66.9(65.1)
Snout to anus 56.4 51.5–57.2(55.2) 55.6–61.2(58.5)
Pre pelvic length 31.9 26.2–34.8(30.5) 28.3–34.9(31.6)
Caudal peduncle length 18.8 18.8–23.1(21.1) 18.4–24.1(21.4)
Caudal peduncle depth 10.6 9.0–12.2(10.6) 9.5–11.4(10.6)
First dorsal-fin base 13.5 10.3–15.2(13.0) 9.9–14.2(11.9)
Second dorsal-fin base 22.3 16.1–22.8(19.1) 14.5–21.2(16.6)
anal fin base 15.2 10.8–15.8(13.8) 10.4–15.7(12.2)
Caudal fin length 28.0 15.4–28.0(22.7) 17.8–23.4(20.3)
Pectoral fin length 25.5 19.3–26.7(22.4) 20.5–21.2(21.0)
Pelvic fin length 11.7 9.6–13.8(11.3) 9.9–12.7(11.5)
Body depth of pelvic fin origin 11.3 9.2–16.3(13.2) 12.0–15.6(14.0)
Body depth of anal fin origin 9.6 9.2–14.6(12.0) 11.3–15.4(12.9)
Pelvic fin origin to anus 25.5 19.3–25.9(22.9) 20.3–26.9(23.5)
Head depth 12.1 10.1–13.8(12.4) 11.3–14.2(13.4)
Percent head length (%)
Snout length 21.1 19.4–30.1(24.6) 15.2–28.6(20.3)
Eye diameter 15.8 11.4–19.5(14.9) 13.1–21.5(17.1)
Cheek depth 23.2 15.2–28.4(24.1) 17.7–25.5(22.2)
Postorbital length 42.1 41.7–54.0(45.7) 45.7–58.2(49.3)
Lower jaw length 26.3 18.8–37.0(29.6) 15.3–25.5(22.7)
Interorbital width 33.7 25.9–39.3(32.4) 26.6–31.6(28.7)
Head width in maximum 49.5 43.3–65.5(54.9) 48.6–64.9(56.5)

Sensory papillae: The sensory papillae arrangement is as follows: Row a ex-

tends to before the middle of the eye. Row b is oblique and reaches forward to
the orbit. Rows c and d extend to the posterior margin of the eyes, and Row cp
is absent. Row f is paired. In the opercular region, there are rows ot, oi, and os.
Rows oi and ot are not connected.
Vertebrae: The total vertebrae count is 12 + 18 = 30 (N = 5) and (P–V) 3/II II
I I 0/9 (N = 5).
Coloration of preserved specimens: In males, the head and body are gray with
irregular markings on the cheeks and operculum. The ventral side is densely cov-
ered with tiny black spots and has six large, sometimes inconspicuous, horizontal
black lines. The first dorsal fin is yellowish, While the second dorsal fin is yellow-

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 8. Dorsal (A), lateral (B), and ventral (C) views of preserved holotype of R. lithopolychroma sp. nov.
(RL20230001 male) and dorsal (D), lateral (E), and ventral (F) views of preserved paratype of R. lithopolychroma sp.
nov. (RL20230101 female).

Figure 9. The skeletal system of R. lithopolychroma sp. nov. Radiograph graphs of the whole body for paratype
RL20230201, females.

ish-brown. The anal fin is yellowish. Females exhibit a gray head and body, with the
first dorsal fin being yellowish and displaying blue spots between the 1st and 2nd
spiny rays. The second dorsal fin is yellowish-brown, and the anal fin is yellowish.
Colour in life: Males display a light gray head and body with irregular orange
markings on the cheeks and operculum, along with three smaller orange lines
along the eyes. The ventral side is densely covered with tiny orange spots and
has six large, sometimes inconspicuous, horizontal black lines. The first dorsal
fin shows orange outlines on spines IV – VII with a white outer edge and blue
spots between the 1st and 2nd spiny rays. The second dorsal fin is orange with
irregular blue markings internally and on the outer edge, as well as blue spots
on the 1st and 2nd spiny rays and a wide white margin. The anal fin is orange at
the base, transitioning to black with a wide white margin. Females also exhibit
a light gray head and body with irregular orange markings on the cheeks and
operculum, and three smaller orange lines along the eyes. The ventral side is
densely covered with tiny orange spots and features six large horizontal black

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 10. Dorsal (A), lateral (B), and ventral (C) views of the head of the preserved holotype of R. lithopolychroma sp.
nov. Red circles indicate sensory canal pores; red dots represent sensory papillae. Abbreviations: AN, anterior nare pore;
PN, posterior nare pore.

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 11. Photographs of R. lithopolychroma captured underwater in a tank A male and B female. Photographed by Mr Zhi.

lines. The first dorsal fin displays orange outlines on spines IV–VII with a yel-
low outer edge and blue spots between the 1st and 2nd spiny rays. The second
dorsal fin is orange, and the anal fin is orange at the base, transitioning to black
with a wide white margin.

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 12. Stream environment in Xiushan, Chongqing where R. lithopolychroma sp. nov.
was collected.

Figure 13. Eggs of R. lithopolychroma sp. nov. at the type locality.

Distribution and habitat. Rhinogobius lithopolychroma is restricted to

fast-flowing, shallow streams with a cobble substrate in Xiushan, Chongqing.
The surveyed streams ranged from 10 to 30 cm in depth. This goby species is
characterized by its large eggs (1.5–2.1 mm in size), which it deposits on the
bottom surface of the cobblestones.
Etymology. Rhinogobius lithopolychroma was discovered in a small stream
with a colorful cobble substrate. Accordingly, we named this species after its

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Lingzhen Li et al.: Two new species of freshwater goby

habitat. In Ancient Greek, “litho” means “stone,” and “polychroma” means rich in
color. We combined these two words to christen this species. We suggest the
Chinese name of this species as “彩石吻虾虎鱼”.

Discussion
Rhinogobius sudoccidentalis and R. lithopolychroma are found in close geo-
graphical proximity and share some environmental commonalities, yet their
morphology differs considerably. Rhinogobius sudoccidentalis typically fea-
tures a longitudinal scale series of 30–33, while R. lithopolychroma exhibits
22–24 scales. In body coloration, R. sudoccidentalis appears creamy white with
black spots on the cheeks and operculum, and a densely spotted ventral side.
Conversely, R. lithopolychroma is light gray with irregular orange markings on
the cheeks and operculum, and a ventral side densely covered with tiny orange
spots, often accompanied by six large, occasionally inconspicuous, horizontal
lines of black.
Morphologically, R. sudoccidentalis bears the closest resemblance to Rhino-
gobius reticulatus Li, Zhong & Wu, 2007 (Fig. 14A, B). They can be distinguished
from other Rhinogobius species by their similar creamy white body coloration,
reddish-brown stripes on the snout, and densely spotted ventral sides. To
differentiate R. sudoccidentalis from R. reticulatus, one should observe traits
such as the absence of predorsal scales in R. sudoccidentalis compared 3–6
in R. reticulatus, and the presence of a lower jaw stripe absent in R. reticulatus.
The closest morphological match to R. lithopolychroma is R. cliffordpopei.
Rhinogobius lithopolychroma and R. cliffordpopei share several distinguishing
characteristics, including VI rays in the first dorsal fin, I,7–8 rays in the anal fin,
and a predorsal scale series count of 0. They also exhibit similar body color-
ation. However, R. lithopolychroma differs from R. cliffordpopei in having 13–15
pectoral fin rays compared to 17–21 in R. cliffordpopei, and a total vertebrae
count of 30 versus 26 in R. cliffordpopei (Li 2011).
As depicted in the phylogenetic tree, R. lithopolychroma is closest to Rhino-
gobius leavelli (Herre, 1935) and Rhinogobius davidi (Sauvage & Dabry de Thier-
sant, 1874), whereas R. sudoccidentalis is closest to Rhinogobius filamentosus
(Wu, 1939), R. wuyanlingensis, R. reticulatus and Rhinogobius duospilus (Herre,
1935) (Fig. 15). Rhinogobius lithopolychroma shares morphological similarities
with R. leavelli and R. davidi, but distinguishes itself with a higher vertebrae
count and a naked predorsal area (Table 4), setting it apart from these species.
Notably, R. sudoccidentalis also exhibits a high vertebrae count compared to
closely related Rhinogobius species, and similarly features a naked predorsal
area and a lower count of longitudinal scale (Table 5). Akihito et al. (2000) sug-
gest that vertebrae counts may correlate with Rhinogobius ecotypes, with spe-
cies inhabiting continental streams and rivers often displaying higher vertebrae
counts (Chen and Miller 2008; Wanghe et al. 2020). The present study supports
this view, noting that R. leavelli, R. davidi, R. filamentosus, R. wuyanlingensis,
R. reticulatus and R. duospilus are primarily found in coastal provinces of south-
ern China (Wu et al. 2008), while both new species are located in inland China.
These two new species represent further evidence of vertebral and environ-
mental adaptations within the genus Rhinogobius.

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 14. Pictures of R. reticulatus and R. sudoccidentalis sp. nov. with the latter having black lines under the eyes
A R. reticulatus B R. sudoccidentalis.

According to studies by Yamasaki et al. (2015) and Li (2011) on Rhinogobius

species, there is a correlation between egg size and species habitat preferences.
Yamasaki defined small eggs as 0.6–0.9 mm and larger eggs as 1.4–2.1 mm.
Li’s research in 2011, conducted in the Qiantang River, demonstrated that spe-
cies like R. duospilus and R. davidi inhabited streams and produced large eggs,
whereas R. similis, typically was found in pond reservoirs and produced small
eggs. Yamasaki et al. (2015) further highlighted that species with small eggs
generally have an amphidromous lifestyle (Takahashi and Yanagisawa 1999;
Keith et al. 2015), while those with large eggs tend to exclusively inhabit streams.

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Lingzhen Li et al.: Two new species of freshwater goby

Figure 15. Maximum likelihood phylogenetic tree for Rhinogobius species having mitochondrial genomes sequences
available, including the two new species highlighted in red.

Table 4. Morphological comparison of Rhinogobius lithopolychroma with the genetically

closest species.

Variable R. lithopolychroma R. leavelli R. davidi

1 dorsal fin
st
VI VI VI
2nd dorsal fin I 9-10 I 8-9 I 9-10
Anal fin I 7-8 I 8-9 I 6-8
Pectoral fin 13-15 14-15 14-15
Longitudinal scale 30-33 28-34 30-32
Transverse scale 7-9 9-11 11-12
Predorsal scale 0 6-12 0-4
Total vertebrae 30 26 28
References This study Wu et al. 2008; Li 2011 Wu et al. 2008; Li 2011

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Lingzhen Li et al.: Two new species of freshwater goby

Table 5. Morphological comparison of Rhinogobius sudoccidentalis with the genetically closest species.

Variable R. sudoccidentalis R. filamentosus R. wuyanlingensis R. reticulatus R. duospilus

1st dorsal fin VI–VII V–VI V–VI VI VI
2 dorsal fin
nd
I 8-9 I 8-9 I 8-9 I 8-9 I 8-9
Anal fin I 6-7 I8 I8 I 7-8 I 6-7
Pectoral fin 14-15 15-17 17-18 15-17 15-16
Longitudinal scale 22-24 30-33 30-32 27-29 30-32
Transverse scale 7-8 8-10 9-10 8-9 8-10
Predorsal scale 0 5-11 7-9 3-6 6-10
Total vertebrae 29 27 27 26-27 27
References This study Wu et al. 2008 Huang et al. 2016 Li et al. 2007 Wu et al. 2008; Li 2011

In the Upper Youshui River catchment, previously documented Rhinogobius

species include R. similis and R. cliffordpopei, known to favor lakes, reservoirs,
and stagnant water environments. Conversely, the new species discovered in
this study exclusively inhabit streams. These newly identified species are all
classified as large-egg types, indicating their better adaptation to stream hab-
itats compared to the small-egg types like R. similis and R. cliffordpopei (Li
2011). Furthermore, the four newly uncovered species exhibit distinct prefer-
ences within stream habitats. For instance, R. lithopolychroma thrives in envi-
ronments characterized by strong currents and low temperatures, specifically
alpine streams with chilly waters, where it represents the predominant Rhinogo-
bius species. On the other hand, R. sudoccidentalis demonstrates a broader dis-
tribution and adaptability, being found in streams with warmer water tempera-
tures, including urban streams. This diversity in habitat preferences suggests
ecological niche differentiation, likely playing a pivotal role in the formation of
Rhinogobius species.
Presently, the survival of the two recently discovered Rhinogobius species
faces certain threats. For instance, manganese ore collection in the headwa-
ters of streams where R. sudoccidentalis resides may have significant implica-
tions for the species survival. Additionally, R. lithopolychroma is restricted to
a narrow habitat and is only found in alpine streams, underscoring the impor-
tance of prioritizing its protection and conducting further detailed studies on its
biology and ecology.

Acknowledgements
We thank Mr Wang from Chongqing and Mr Wu from Yunnan for their help
in sample collection. Thanks to Mr Zhi and Mr Luo for providing the photos.
Thanks to Mr Wang of CAS for providing the filming and scanning equip-
ment. Thank you to Mr Shao and Mr Zhou for their extensive revisions of
the paper.

Additional information
Conflict of interest
The authors have declared that no competing interests exist.

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Lingzhen Li et al.: Two new species of freshwater goby

Ethical statement
No ethical statement was reported.

Funding
National Talent Research Grant for 2023 (No. 5330500953);Natural Science Foundation
of Chongqing (Grant No. CSTB2022NSCQ-MSX0566).

Author contributions
Lingzhen Li: methodology, formal analysis, validation, writing-original draft, writing-re-
view, editing, investigation. Chaoyang Li: methodology, investigation, formal analysis,
formal analysis. Weihan Shao: data curation, project administration, resources, super-
vision, writing-review and editing. Suxing Fu: data curation, project administration, re-
sources, supervision, writing-review and editing. Chaowei Zhou: data curation, project
administration, resources, supervision, writing-review and editing.

Author ORCIDs
Lingzhen Li https://orcid.org/0009-0008-2139-4009
Suxing Fu https://orcid.org/0009-0001-0562-1469

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

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