Description of a new troglobitic Sinocyclocheilus (Pisces, Cyprinidae) species from the upper Yangtze River Basin in Guizhou, South China

Sinocyclocheilus guiyang , a new troglobitic species from a subterranean tributary of the upper Yangtze Basin in Guiyang City, Guizhou Province, China is described in the present study. The new species is distinguishable from its congeneric species by a combination of the following characters: tip of maxillary barbel reaching to posterior edge of orbit; forehead horn absent; eye absent (or highly reduced) and tip of pectoral fins not significantly extending beyond the base of the pelvic fin. Molecular evidence, based on the mitochondrial cytochrome b (cyt b ) gene, further supports the validity of the species and also reveals its close relationship with S. cyphotergous , S. multipunctatus , S. punctatus and S. sanxiaensis . In addition, the new species faces a high risk of extinction, underscoring the urgency for habitat protection measures within its limited range.


Introduction
Sinocyclocheilus Fang, 1936 (golden-line barbel), endemic to south China, is one of the most diversified genera in the family Cyprinidae, consisting of more than seventy species (Jiang et al. 2019;Mao et al. 2022;Xu et al. 2023;Luo et al. 2024).Highly-developed subterranean river systems in this region are a major contributor to its remarkable diversity because Sinocyclocheilus is restricted to subterranean river systems and adjacent regions (Ma et al. 2019).To date, sixty-six Sinocyclocheilus species are endemic to the Pearl River Basin, with only six species (S. grahami Regan, 1904, S. wumengshanensis Li, Mao & Lu, 2003, S. huizeensis Cheng, Pan, Chen, Li, Ma & Yang, 2015, S. wui Li & Li, 2013, S. sanxiaensis Jiang, Li, Yang & Chang, 2019and S. multipunctatus Pellegrin, 1931) occurring in the Yangtze River Basin (Eschmeyer et al. 2024).In addition, three of them (S.grahami, S. wumengshanensis and S. huizeensis) are restricted to Yunnan Province which belongs to the Jinshajiang River (a section of the mainstream of upper Yangtze River) System.Only one species, S. multipunctatus, is found in the Wujiang River System, a major southern tributary of the upper Yangtze River in Guizhou Province.
However, similar to the Pearl River Basin, the Wujiang River System also exhibits extensive and well-developed karst landforms (Che and Yu 1985), which has provided good conditions for the formation of subterranean river systems and the subsequent evolution of troglobitic fishes.This can be seen in the high diversity of the hypogean Triplophysa which has five described species within this river system (Liu et al. 2022).Moreover, a recent breakthrough study reported S. sanxiaensis from the Three Gorges Reservoir which belongs to the mainstream of the upper Yangtze River Basin in west Hubei Province.Molecular phylogenetic analysis has grouped S. sanxiaensis with S. cyphotergous Dai, 1988, S. multipunctatus and S. punctatus Lan & Yang, 2017(Jiang et al. 2019).The S. cyphotergous -S.multipunctatus group, herein defined for the abovementioned four species, is characterised by a typically convex dorsal profile, short barbels and high head depth.For a long time, members of this species group were only mainly known from Hongshui River (a section of the mainstream of the Pearl River) System and Liujiang River (north tributary of the Pearl River) System, with only S. multipunctatus spanning from Hongshui River to Wujiang River (Wu 1989;Zhao and Zhang 2009).The description of S. sanxiaensis greatly expanded the distribution boundary of this species group, providing insights into the potentially underestimated diversity of Sinocyclocheilus in the Wujiang River System, situated between the Three Gorges Reservoir and the Hongshui River.
Various morphological features that are adapted to subterranean environments have been found in troglobitic species of Sinocyclocheilus including degenerated eyes, reduced (or lost) pigmentation, degenerated scales, elongated fins and horn-like structures (e.g.humpback and horn) (Zhao and Zhang 2009;Ma et al. 2019).In addition, possession of extended barbels in Sinocyclocheilus species is common as the long barbels better detect water flow and aid foraging in subterranean water systems which are marked by permanent darkness and food scarcity (Ma et al. 2019).Fewer than one third (twenty-one) of Sinocyclocheilus species have short maxillary barbels that do not extend to the posterior edge of the preoperculum (Zhao and Zhang 2009;Lan et al. 2013;Xu et al. 2023) and more than half possess horn-like structures (e.g. S. anatirostris Lin & Luo, 1986, S. aquihornes Li & Yang, 2007, S. cyphotergous, S. rhinocerous Li & Tao, 1994and S. longicornus Luo, Xu, Wu, Zhou & Zhou, 2023).Amongst the currently recognised species of Sinocyclocheilus, only two, S. jinxiensis Zheng, Xiu & Yang, 2013 and S. sanxiaensis, possess a combination of short maxillary barbels, degenerated eyes, reduced pigmentation and lack of horn-like structures.These combined characters are unique within Sinocyclocheilus and represent exceptional cases for evolutionary studies.
The authors conducted a fish field survey in a subterranean stream within the Wujiang River System in central Guizhou Province, south China.This survey yielded seven specimens characterised by short maxillary barbels, no horn-like structures and absent or highly reduced eyes, traits shared with S. jinxiensis and S. sanxiaensis.Careful morphological examination revealed that they are, in fact, not conspecific with any other known species of Sinocyclocheilus and, thus, represent an unnamed species.Genetic analyses further revealed that these specimens formed a distinct cluster within the S. cyphotergous -S.multipunctatus group.The purpose of the present paper is to provide a formal description of this unnamed species, based on multiple lines of evidence including morphological and molecular datasets.

Specimen sampling and preservation
The treatment of experimental animals in this study was consistent with the Chinese animal welfare laws (GB/T 35892-2018).Specimens were collected from central-south Guizhou and north Guangxi from 2019 to 2023.After anaesthesia, specimens were fixed in 10% formaldehyde and then preserved in 75% ethanol for morphological comparison.The right pelvic fin of some specimens was dissected and fixed in 95% ethanol for DNA extraction.Specimens newly collected for this study have been deposited in the Institute of Hydrobiology, Chinese Academy of Sciences (IHB), Guangxi University (GXU) and Zhejiang Forest Resource Monitoring Center (ZJFR).Other comparative materials have been stored in Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Morphological analyses
Measurements were taken point-to-point on the left side of the specimens with a Vernier caliper to a precision of 0.1 mm.All measurements, counts and terminologies follow Zhao et al. (2006), with the following exceptions: maxillary barbel in the present study refers to the barbel rooted at the corner of the mouth and rostral barbel refers to the barbel rooted at the rostrum.The major morphometrics are shown in Fig. 1.All morphometric measurements have been transferred to percentage of standard length (SL) and recorded to the closest 0.1%.The caudal peduncle depth (CPD) to caudal peduncle length (CPL) ratios are calculated and recorded to the closest 0.01.For osteological observation, specimens were scanned by micro-computed tomography (micro-CT) (Siemens Somatom Definition X-ray machine).The 3D renderings of the osteological structure of the whole specimen and pharyngeal dentition were created and visualised in VG Studio Max 2.1 (He et al. 2013).Total vertebrae were counted from the first free vertebra to the last half-centrum.
Morphometric measurements were subject to principal component analysis (PCA) to explore the relative contributions of specific variables to morphological variations.PCA was conducted on the Statistical Package for the Social Sciences (SPSS) 19.0 (IBM, Armonk, NY, USA).Prior to PCA, all included measurements were normalised by log transformation.Linear regression analysis for origin data of each character was also computed on SPSS 19.0.

DNA extraction, PCR and sequencing
Genomic DNA was extracted from 95% ethanol-fixed fin tissue using the modified salt-extraction method described by Tang et al. (2008).Fragments containing the mitochondrial cytochrome b (cytb) gene were amplified by polymerase chain reaction (PCR) with the primer pairs (L14724 and H15915) (Zhao et al. 2006) in a 30 μl reaction system: 3 μl 10 × PCR buffer, 30-50 ng DNA template, 1 μl primers (each 10 μM), 1.5 μl dNTPs (each 2.5 mM), 2.5 U Taq DNA polymerase and ddH 2 O added to reach the final volume.PCR procedures also follow Tang et al. (2008).The PCR products were purified and sequenced in both directions with the corresponding primers by a commercial sequencing company.All newly-generated sequences have been submitted to GenBank.

Molecular data analyses
Phylogenetic analysis was performed, based on nine newly-obtained cytb sequences and an additional 47 sequences downloaded from NCBI GenBank, including 49 Sinocyclocheilus species and a single species Cyprinus carpio Linnaeus, 1758 as outgroup (Table 1).The sequences were revised manually and then aligned using Clust-alW in MEGA7.0 (Kumar et al. 2016).Both Maximum Likelihood (ML) and Bayesian Inference (BI) methods were utilised to reconstruct the phylogenetic relationship.The optimal nucleotide substitution model was selected in ModelFinder (Kalyaanamoorthy et al. 2017) according to Akaike Information Criterion.Maximum Likelihood analysis was run in IQ-TREE 1.6.8(Nguyen et al. 2015), with the selected TIM3+F+I+G4 model and 1,000 non-parametric bootstrap replicates.Bayesian Inference was performed in MrBayes 3.2.6 (Ronquist et al. 2012) under the selected GTR+F+I+G4 model, using the MCMC method (four chains simultaneously run for 20,000,000 generations) to calculate posterior probability, with tree sampling frequency set to 1 per 1000 cycles and the initial 25% of the sampled data discarded as burnin.The convergence of BI analysis was reached when the average standard deviation of split frequencies was less than 0.01.Uncorrected pairwise genetic distances (p-distance), based on cytb, were computed in MEGA 7.0.
The major diagnostic characters for S. guiyang and related species are summarised in Table 3. Description.Morphometric measurements of type specimens have been transferred to percentage of standard length (SL), as summarised in Table 2. Body laterally compressed; maximum body depth positioned at insertion of dorsal-fin.Dorsal profile convex from snout tip to dorsal-fin base end and slightly concave after dorsal-fin base.Ventral profile of pre-anal part slightly convex and slightly concave after anal-fin origin.
Head slightly compressed, conical in lateral view.Eyes absent (5) or highly reduced and partially covered with skin (2).Eye orbits located in dorsal anterior part of head, filled with soft tissue.Nostrils located at midway between snout tip and anterior margin of orbit; anterior nostril with rim forming an oblique tube, posteriorly thickening and elongating; posterior nostril open and elliptical.Snout blunt in dorsal view and slightly pointed in lateral view.Mouth subterminal and arched; with two pairs of barbels; rostral pair positioned anterior to anterior nostril, extend-ing to the insertion of anterior margin of orbit, being 6.2% (4.9-7.1%) of SL; maxillary pair positioned at corners of mouth, extending to the posterior margin of orbit, being 7.1% (5.8-8.3%) of SL.Gill opening large; opercular membranes not connected at isthmus.Joints of dentary-angulars not close at isthmus.Ten outer rakers (1) on first gill arch.Pharyngeal teeth pattern 1,3,4-4,3,0 (1); tooth tip pointed and compressed.Vertebrae 36 (2) (Fig. 2C).
All original morphometric measurements and meristic counts are available in Suppl.material 1.
Colouration.In freshly collected individuals (Figs 2D, 3), head and body generally pinkish, with or without pigments dorsally.A pair of dark stripes present on dorsal-posterior part of head, extending to dorsal mid-point of nape; a gold stripe extending along dorsal mid-line from nape to dorsal-fin origin.All fins transparent.
In preserved specimens (Fig. 2A, B), body and head slightly yellowish, with or without pigments dorsally.Abovementioned dark stripes and gold stripe faded.All fins transparent.
Distribution and habitat.This species is presently only known from a subterranean stream flowing into the Wujiang River in the upper Yangtze River Basin in Qingzhen County, Guiyang City, Guizhou Province, China (Fig. 4).The species inhabits pools of subterranean stream with gravel substrate (Fig. 5).Video record of Sinocyclocheilus guiyang in situ is available in Suppl.material 2.
Etymology.The location of the subterranean stream where this new species was first collected: Guiyang City, the capital of Guizhou Province, is directly utilised as a specific epithet.The common name proposed for the new species is '贵阳金线鲃' (Guiyang Golden-line Barbel).
Morphometric comparisons.Principal component analysis for Sinocyclocheilus guiyang, S. punctatus, S. multipunctatus and S. sanxiaensis, based on 29 log-transformed characters, showed that 95.23% of total variance was explained by the first three components, including 87.24% by PC1, 4.79% by PC2 and 3.20% by PC3, respectively.In the PC1 vs. PC3 scatter plot, S. guiyang and S. punctatus form a distinct cluster from the other two congeners on the PC3 axis (Fig. 6A).The characters with major loading on PC3 included maxillary barbel length, rostral barbel length, eye diameter, width between posterior nostrils and pectoral-fin base length (Table 4).Further PCA in S. guiyang and S. punctatus demonstrated that the first three components explained 97.26% of total variance, in which PC1, PC2 and PC3 explained 93.31%, 2.29% and 1.66%, respectively.Sinocyclocheilus guiyang is separated from S. punctatus on the PC2 axis in the PC1 vs. PC2 scatter plot (Fig. 6B).Eye diameter, maxillary barbel length, width between posterior nostrils, rostral barbel length and snout width are major loading characters on PC2 (Table 4).Linear regression analysis also support S. multipunctatus as distinct from S. guiyang and S. punctatus by shorter maxillary  and rostral barbel lengths (6.0-10.7%SL vs. 4.9-7.1% in S. guiyang, 4.9-  6.7% in S. punctatus) (Fig. 6C, D), whereas S. guiyang further differed from S. punctatus by shorter prenostril length (4.5-6.0% of SL vs. 5.8-7.6%) and higher caudal peduncle depth to caudal peduncle length ratio (0.55-0.67 vs. 0.45-0.56)(Fig. 6E, F).Molecular data analyses.A total of 1134 bps were included in the aligned dataset of cytb gene, with 661 conservative sites, 473 variable sites, 390 parsimony informative sites and 83 singleton sites.The mean frequency of four nucleotides in the sequences of Sinocyclocheilus guiyang is A = 29.7%,G = 14.2%,C = 26.2% and T = 29.9%.The phylogenetic trees, reconstructed by ML and BI methods, are identical in topology (Fig. 7).The monophyletic linage of Sinocyclocheilus guiyang is robustly supported by 100% posterior probabilities and 99% bootstrap supports and is sister to S. punctatus.The lineage of the two spe- cies clustered with the lineage comprising sequences of S. multipunctatus, S. cyphotergous and S. sanxiaensis.Additionally, the topology of phylogenetic reconstruction in the present study supports the monophyly of the S. cyphotergous -S.multipunctatus species group.Average genetic distances derived from cytb sequences of Sinocyclocheilus species distributed in Guizhou Province or the Yangtze River Basin are given in Table 5.The intraspecific distance of S. guiyang is 0.1% and the mean distances between the new species and other congeners range from 2.3% (vs.S. punctatus) to 13.8% (vs.S. wumengshanensis).

Discussion
The new species is the first described troglobiotic species of genus Sinocyclocheilus in the Wujiang River of upper Yangtze River Basin in Guizhou Province.Three characters are useful for distinguishing Sinocyclocheilus guiyang from all other Sinocyclocheilus species, except S. jinxiensis and S. sanxiaensis (Fig. 8): short maxillary barbel not reaching posterior edge of preoperculum, absence of horn-like structure and degenerated (lost or highly reduced) eye.It is distinct from S. jinxiensis and S. sanxiaensis in having shorter (vs.longer) pectoral fins, just reaching (vs.significantly extending beyond) the base of pelvic fin.It further differs from S. jinxiensis in possessing degenerated body scales (20-24 scale rows above lateral line vs. 8-9), and from S. sanxiaensis in having shorter (vs.longer) snout (length 9.9-11.0% of SL vs. 16.4%) and longer (vs.shorter) maxillary barbel, reaching to posterior edge of orbit, length 5.8-8.3% of SL (vs.not reaching to anterior edge of orbit, 4.2%).Wang & Liao, 1997, a cave-dwelling species, also demonstrated polymorphism in eye mode (Wen et al. 2023) Sinocyclocheilus guiyang is undoubtedly a member of the S. cyphotergous -S.multipunctatus species group, as evidenced by both morphology and phylogenetic results in this study.The monophyly of the S. cyphotergous -S.multipunctatus group has also been confirmed in previous works (Wen et al. 2022;Jiang et al. 2023;Xu et al. 2023).Excluding S. sanxiaensis, which has been mentioned above, S. guiyang differs from S. cyphotergous by the absence of a humpback, from S. multipunctatus in having developed pectoral fins extending (vs.not extending) to pelvic-fin insertion and fewer lateral line scales (45-47 vs. 53-60), from S. punctatus in having longer maxillary barbels reaching to posterior edge of orbit (vs.reaching to anterior edge of orbit).The principal component analysis of measurement characters also confirms the morphological distinctness of S. guiyang from other members of the S. cyphotergous -S.multipunctatus group lacking horn-like structures (Fig. 6A).
As sister to one another, the pairwise distance between Sinocyclocheilus guiyang and S. punctatus is 2.3%, exceeding the 2% mitochondrial DNA threshold which is often indicative of valid species in most groups (Avies and Walker 1999) and coincides with distances used for currently described species in Sinocyclocheilus.The genetic distances between sibling species being comparatively low might be a trait common across the genus Sinocyclocheilus, which has undergone recent divergence in extreme subterranean environments (Mao et al. 2022).In addition, S. guiyang and S. punctatus display allopatric distribution patterns as S. punctatus is confined in the Longjiang River (a tributary of Liujiang River in the Pearl River Basin), while S. guiyang only occurs in Wujiang River that belongs to the upper Yangtze River Basin.The species validity of S. guiyang can be confirmed under an integrative framework combining numerous lines of evidence, comprised of morphological distinctness, molecular phylogeny and geographical range.Moreover, samples of S. multipunctatus formed a paraphyletic entity in the phylogenetic tree.It is evident that geographic divergence has occurred as the Yangtze River population showed close affinities to S. sanxiaensis, while the Pearl River population is located at the base of the paraphyly (Fig. 7), implying the existence of cryptic species diversity within S. multipunctatus that warrants taxonomic revision in the future.
The fish diversity in the Yangtze River Basin of Guiyang City, the most urbanised area of Guizhou, has long been underestimated (Zeng and Liu 2020).The increasing discoveries of narrowly distributed species in this area have raised concerns for the conservation of these species  (Liu et al. 2022;Zeng et al. 2022).Human disturbance to the habitat and climate change have exacerbated the risk of extinction of cave species (Shu et al. 2013;Mouser et al. 2022).Recent years have witnessed a dramatic increase in land use, fishing pressure, cave tourism and invasion of a large amount of Procambarus clarkii Girard, 1852 in the type locality of Sinocyclocheilus guiyang (See Suppl.material 4), as well as frequent onset of droughts.Moreover, the estimated population size of the new species is extremely small, as only 25 individuals in total were recorded during our surveys and has suffered a population decline in the recent 2 years (Table 6).S. guiyang will be automatically assigned to the list of 2 nd Class of the national protected animals after its description as the whole Sinocyclocheilus species are within this list since 2021.Construction of a small conservation area with limited human disturbance in the existing habitats of this new species under extreme threat should be considered in priority.
Sinocyclocheilus multipunctatus: IHB 2014040001, 1 specimen, 96.4 mm SL; a vauclusian spring of the Mengjiang River System in the Pearl River Basin at

Figure 3 .
Figure 3. Intraspecific morphological variations of Sinocyclocheilus guiyang. A. Individual with no eyes; B. Individual with highly reduced eyes, partially covered with skin; C. Individual with dorsal pigment; D. Individual without pigment.Note that individuals of both colouration types share the presence of dark stripes on the dorsal-posterior part of the head and a gold stripe along the midline from the nape to the dorsal-fin origin.

Figure 4 .
Figure 4. Sampling sites of Sinocyclocheilus guiyang and related species in this study.

Figure 5 .
Figure 5. Habitat of Sinocyclocheilus guiyang. A. The pool of a subterranean stream where S. guiyang was collected; B. S. guiyang in situ.

Figure 6 .
Figure 6. A. Scatter plot of 1 st and 3 rd principal components for Sinocyclocheilus guiyang, S. punctatus, S. multipunctatus and S. sanxiaensis; B. Scatter plot of 1 st and 2 nd principal components for S. guiyang and S. punctatus; relationships between C. Rostral barbel length and head length, D. Maxillary barbel length and head length for S. guiyang, S. punctatus and S. multipunctatus; relationships between E. Prenostril length and head length, F. Caudal peduncle depth and caudal peduncle length for S. guiyang and S. punctatus.

Figure 7 .
Figure 7. Phylogenetic tree of Sinocyclocheilus species inferred from cytb using Bayesian Inference and Maximum Likelihood methods.Node values show posterior probabilities/bootstrap supports if greater than 50%.Currently known distribution of species of the S. cyphotergous -S.multipunctatus group are shown on map.

Table 1 .
GenBank accession numbers for molecular phylogenetic analysis.n/a, not available.

Table 3 .
Major diagnostic characters for Sinocyclocheilus guiyang and its close congeners.n/a, not available.

Table 4 .
PCA loadings of the first three principal components extracted from 29 morphometric data for Sinocyclocheilus guiyang and related species.

Table 5 .
Average uncorrected pairwise genetic distance (p-distance, %) derived from cytb in 15 species of Sinocyclocheilus distributed in Guizhou Province or the Yangtze River Basin.Bold numbers, intraspecific distances; regular numbers, interspecific distances; n/a, not available. .
netic relationships with S. guiyang, which all belong to the S. cyphotergous -S.multipunctatus group.Sinocyclocheilus guiyang can be further differentiated from S. jiuxuensis, S. brevibarbatus and S. mashanensis by the following

Table 6 .
Number of individuals of Sinocyclocheilus guiyang in type locality recorded each year during the survey.