﻿Two new species of freshwater goby (Teleostei, Gobiidae) from the Upper Youshui River, Chongqing, China

﻿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 Rhinogobiussudoccidentalis and Rhinogobiuslithopolychroma. 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. Rhinogobiussudoccidentalissp. nov. is characterized 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. Rhinogobiuslithopolychromasp. 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 vertebral count is 30, with 12 precaudal and 18 caudal vertebrae. The head and body of this species are light gray with irregular orange markings on the cheeks and opercle. Through morphological and molecular analyses, it has been confirmed that R.lithopolychroma and R.sudoccidentalis represent novel species within the Rhinogobius genus.


Introduction
The genus Rhinogobius, belonging to the subfamily Gobionellinae within the family Gobiidae, is widely distributed across East and Southeast Asia.First described 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 described, with an increasing number of new species being discovered.In recent years, several new species of Rhinogobius have been found in China, including R. houheensis Kunyuan et al., 2020, R. coccinella Endruweit, 2018, R. maculagenys 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., 2022and 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 underestimated.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 encompasses numerous stream habitats within its mountainous areas.The biodiversity in Xuan'en and Fanjingshan, traversed by the Upper Yuanjiang River Basin, is exceptionally high and potentially serves as a glacial refuge (Fei et al. 2017).Consequently, 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 Chongqing, two species of Rhinogobius were discovered.Historically, only R. similis and Rhinogobius cliffordpopei (Nichols, 1925) were documented in the Yuanjiang 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 Rhinogobius in the Upper Youshui River to explore the habitat segregation of Rhinogobius, building upon previous research concerning the ecological preferences of Rhinogobius species (Sone et al. 2001;Ito et al. 2006).

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 pterygiophores and neural spines (Akihito et al. 1984).For example, in the formula (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 3 rd and 4 th vertebrae; 2 between the neural spine of the 4 th and 5 th vertebrae; 1 between the neural spine of the 5 th and 6 th vertebrae; and 1 between the neural spine of the 5 th and 6 th vertebrae; "0" indicates no pterygiophore between the neural spines of the 7 th and 8 th vertebra; "9" indicates that the first pterygiophore of the 1 st ray of the 2 nd dorsal fin is inserted above the 9 th 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 Nanoball) 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.Complete mitochondrial genomes were aligned using MAFFT v.7.244 (Katoh and Standley 2013).The obtained mitochondrial gene was compared with Rhinogobius 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 using maximum likelihood (ML) methods with RAxML v. 8.2.3 (Stamatakis 2014), incorporating mitochondrial gene data from the GenBank library (Table 1).Outgroup specimens were analyzed using Tridentiger kuroiwae Jordan & Tanaka, 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.

Morphological analyses
Rhinogobius sudoccidentalis sp.nov.https://zoobank.org/975F33AC-F810-4F32-8D57-26A583D924BBDiagnosis.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 extends to the mandible.Morphometrics Reference Table 2.
Description.Fins: The fins display distinct features: the first dorsal fin typically bears VI rays (18) or VII rays (2), while the second dorsal fin exhibits either I, 8 rays (2) or I, 9 rays (18).The 3 rd or 4 th 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 1 st or 2 nd 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 2 nd and 3 rd 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-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.Preopercular 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.

Coloration of preserved specimens:
In males, the head and body of R. sudoccidentalis 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-   tling pattern between the 1 st and 2 nd 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 reddish 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.Distribution and habitat.Rhinogobius sudoccidentalis was initially discovered 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 populations of this species were also observed in Tongren, Guizhou Province.In the Xiushan area, R. sudoccidentalis is the dominant fish species, utilizing the 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-F9DBAD7F197ADiagnosis.Rhinogobius lithopolychroma can be distinguished from other species in the Rhinogobius by the following characteristics: It typically possesses 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 depressed, the rear tip extends to the base of the second branched ray of the second 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 occipital 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 posterior edge of eyes and a lack of ω1.The lateral section of anterior oculoscapular canal includes pores α and a terminal pore ρ.The posterior oculoscapular canal possesses two terminal pores θ and τ.Preopercular canals are presented, with pores ε, γ, and δ.

Sensory papillae:
The sensory papillae arrangement is as follows: Row a extends 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.

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 covered 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-  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 1 st and 2 nd 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 1 st and 2 nd 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 1 st and 2 nd 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

A B
lines.The first dorsal fin displays orange outlines on spines IV-VII with a yellow outer edge and blue spots between the 1 st and 2 nd 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.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 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 geographical proximity and share some environmental commonalities, yet their morphology differs considerably.Rhinogobius sudoccidentalis typically features 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 Rhinogobius 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 coloration.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 Rhinogobius leavelli (Herre, 1935) and Rhinogobius davidi (Sauvage & Dabry de Thiersant, 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) suggest that vertebrae counts may correlate with Rhinogobius ecotypes, with species 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 southern China (Wu et al. 2008), while both new species are located in inland China.These two new species represent further evidence of vertebral and environmental adaptations within the genus Rhinogobius.

A B
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 0.6-0.9mm and larger eggs as 1.4-2.1 mm.Li's research in 2011, conducted in the Qiantang River, demonstrated that species 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.

References
This study Wu et al. 2008;Li 2011Wu 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 habitats compared to the small-egg types like R. similis and R. cliffordpopei (Li 2011).Furthermore, the four newly uncovered species exhibit distinct preferences within stream habitats.For instance, R. lithopolychroma thrives in environments characterized by strong currents and low temperatures, specifically alpine streams with chilly waters, where it represents the predominant Rhinogobius species.On the other hand, R. sudoccidentalis demonstrates a broader distribution and adaptability, being found in streams with warmer water temperatures, 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 headwaters of streams where R. sudoccidentalis resides may have significant implications for the species survival.Additionally, R. lithopolychroma is restricted to a narrow habitat and is only found in alpine streams, underscoring the importance of prioritizing its protection and conducting further detailed studies on its biology and ecology.

Figure 1 .
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.

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

Figure 4 .
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

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

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

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

Figure 10 .
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.

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

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

Figure 14 .
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.

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

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

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

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

Table 4 .
Morphological comparison of Rhinogobius lithopolychroma with the genetically closest species.

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