Zoosyst. Evol. 100 (2) 2024, 515-529 | DOI 10.3897/zse.100.119520 Gee Ee BERLIN Description of a new troglobitic Sinocyclocheilus (Pisces, Cyprinidae) species from the upper Yangtze River Basin in Guizhou, South China Wei-Han Shao!, Guang- Yuan Cheng?, Xiao-Long Lu®, Jia-Jun Zhou*, Zhi-Xuan Zeng® Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Guiyang Bureau of Ecology and Environment, Guiyang, China Guiyang Oianren Ecological Conservation Center, Guiyang, China Zhejiang Forest Resource Monitoring Center, Hangzhou, China Zhejiang Forestry Survey Planning and Design Company Limited, Hangzhou, China Dn oO FP WDNY The Department of Endocrinology, Branch of National Clinical Research Center for Metabolic Diseases, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China https://zoobank. org/5B410772-DA79-437F-SABB-A047B4FDC604 Corresponding authors: Zhi-Xuan Zeng (985801524@qq.com); Jia-Jun Zhou (cnwaters@foxmail.com) Academic editor: Nicolas Hubert # Received 29 January 2024 # Accepted 28 March 2024 Published 14 May 2024 Abstract 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 6 (cytb) 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. Key Words cavefish, conservation, morphology, phylogenetic analysis, Yangtze River Introduction Sinocyclocheilus Fang, 1936 (golden-line barbel), endem- ic 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 re- markable 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, 2019 and S. multipunctatus Pellegrin, 1931) occurring in the Yangtze River Basin (Eschmey- er 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) Sys- tem. Only one species, S. mu/tipunctatus, is found in the Wuyiang 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 Copyright Shao, W.-H. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 516 karst landforms (Che and Yu 1985), which has provided good conditions for the formation of subterranean river systems and the subsequent evolution of troglobitic fish- es. 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 break- through study reported S. sanxiaensis from the Three Gorges Reservoir which belongs to the mainstream of the upper Yangtze River Basin in west Hubei Province. Mo- lecular 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, 1s 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 Liuji- ang River (north tributary of the Pearl River) System, with only S. multipunctatus spanning from Hongshui River to Wuyiang River (Wu 1989; Zhao and Zhang 2009). The description of S. sanxiaensis greatly expanded the distri- bution boundary of this species group, providing insights into the potentially underestimated diversity of Sinocy- clocheilus 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, elon- gated fins and horn-like structures (e.g. humpback and horn) (Zhao and Zhang 2009; Ma et al. 2019). In addi- tion, possession of extended barbels in Sinocyclocheilus species 1s common as the long barbels better detect wa- ter flow and aid foraging in subterranean water systems which are marked by permanent darkness and food scar- city (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 preoper- culum (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, 1994 and S. longicornus Luo, Xu, Wu, Zhou & Zhou, 2023). Amongst the currently recognised species of Sino- cyclocheilus, only two, S. jinxiensis Zheng, Xiu & Yang, 2013 and S. sanxiaensis, possess a combination of short maxillary barbels, degenerated eyes, reduced pigmen- tation and lack of horn-like structures. These combined characters are unique within Sinocyclocheilus and repre- sent exceptional cases for evolutionary studies. The authors conducted a fish field survey in a subterra- nean stream within the Wuyiang River System in central Guizhou Province, south China. This survey yielded sev- en 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 mor- phological examination revealed that they are, in fact, not zse.pensoft.net Shao, W.-H. et al.: A new troglobitic Sinocyclocheilus species from China conspecific with any other known species of Sinocyclochei- /us and, thus, represent an unnamed species. Genetic analy- ses 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 de- scription of this unnamed species, based on multiple lines of evidence including morphological and molecular datasets. Material and methods 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 cen- tral-south Guizhou and north Guangxi from 2019 to 2023. After anaesthesia, specimens were fixed in 10% formal- dehyde and then preserved in 75% ethanol for morpho- logical comparison. The right pelvic fin of some speci- mens 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 (THB), 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 morpho- metrics are shown in Fig. 1. All morphometric measure- ments 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 mi- cro-computed tomography (micro-CT) (Siemens Soma- tom Definition X-ray machine). The 3D renderings of the osteological structure of the whole specimen and pharyn- geal 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 contri- butions of specific variables to morphological variations. PCA was conducted on the Statistical Package for the So- cial Sciences (SPSS) 19.0 IBM, Armonk, NY, USA). Pri- or 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. Zoosyst. Evol. 100 (2) 2024, 515-529 ou, Figure 1. Major morphometrics demonstrated on original drawing of Sinocyclocheilus guiyang. standard length (1), head length (2), eye diameter (3), snout length (4), pre-nostril length (5), head depth (6), body depth (7), caudal peduncle depth (8), upper jaw length (a—c), lower jaw length (b—c), pre-pectoral length (a—d), pre-dorsal length (a—f), pre-pelvic length (a—h), pre-anal length (a—m), pectoral-fin base length (d—e), pectoral-fin length (d—g), dorsal-fin length (f-j), dorsal-fin base length (f-1), pelvic-fin base length (h-i), pelvic-fin length (h—k), anal-fin base length (m—n), anal-fin length (m—o), caudal peduncle length (n—p). DNA extraction, PCR and sequencing Genomic DNA was extracted from 95% ethanol-fixed fin tissue using the modified salt-extraction method de- scribed 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 ul reaction system: 3 ul 10 x PCR buffer, 30-50 ng DNA template, 1 ul primers (each 10 uM), 1.5 wl dNTPs (each 2.5 mM), 2.5 U Taq DNA polymerase and ddH,O add- ed to reach the final volume. PCR procedures also fol- low Tang et al. (2008). The PCR products were purified and sequenced in both directions with the corresponding primers by a commercial sequencing company. All new- ly-generated sequences have been submitted to GenBank. Molecular data analyses Phylogenetic analysis was performed, based on nine new- ly-obtained cytb sequences and an additional 47 sequenc- es downloaded from NCBI GenBank, including 49 Sino- cyclocheilus 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) accord- ing to Akaike Information Criterion. Maximum Like- lihood 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 In- ference 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 probabili- ty, with tree sampling frequency set to 1 per 1000 cycles and the initial 25% of the sampled data discarded as burn- in. 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-dis- tance), based on cytb, were computed in MEGA 7.0. Results Sinocyclocheilus guiyang sp. nov. https://zoobank. org/67337E27-2D9 1-4C21-B557-AA36ECABDAAI1 Fig. 2, Table 2 Type materal. Holotype. [HB 202012250001, 124.0 mm SL; China: Guizhou Province: Guiyang City: Qingzhen County: a subterranean stream tributary of the Wujiang System in the upper Yangtze River Basin, 26°50'26"N, 106°16'37"E, 1250 m elevation; Jia-Jun Zhou, Dec 2020. Paratypes. THB 201911140001, 1 specimen, 57.5 mm SL; Zhi-Xuan Zeng, Nov 2019; other data same as ho- lotype. IHB 202012250002, 1 specimen, 86.4 mm SL; collected with holotype. THB 202207260001, GxXU 202207260002—04, 4 specimens, 124.3-174.1 mm SL; Jia-Jun Zhou, Jul 2022; other data same as holotype. Diagnosis. Sinocyclocheilus guiyang 1s distinguish- able from all other congeners by a combination of the zse.pensoft.net 518 Shao, W.-H. et al.: A new troglobitic Sinocyclocheilus species from China Table 1. GenBank accession numbers for molecular phylogenetic analysis. n/a, not available. Taxon Voucher specimen Locality Accession No. Source Sinocyclocheilus sanxiaensis KIZ 2019000001 Hubei, Yangtze River MN106258 NCBI Sinocyclocheilus grahami XHO701 Yunnan, Yangtze River AY854694 NCBI Sinocyclocheilus wumengshanensis YNUSM2016081 7008 Yunnan, Yangtze River MGO021442 NCBI Sinocyclocheilus guiyang 01 IHB 202012250001 Guizhou, Yangtze River OR141734 This study Sinocyclocheilus guiyang 02 IHB 202012250002 Guizhou, Yangtze River OR141735 This study Sinocyclocheilus guiyang 03 IHB 202207260001 Guizhou, Yangtze River OR141736 This study Sinocyclocheilus multipunctatus 01 IHB 202302080001 Guizhou, Yangtze River OR141737 This study Sinocyclocheilus multipunctatus 02 n/a Guizhou, Pearl River MG026730 NCBI Sinocyclocheilus cyphotergous 01 IHB 202302080002 Guizhou, Pearl River OR141738 This study Sinocyclocheilus cyphotergous 02 IHB 202207280010 Guizhou, Pearl River OR141739 This study Sinocyclocheilus punctatus 01 ZJFR 2311001 Guangxi, Pearl River PP112594 This study Sinocyclocheilus punctatus 02 ZJFR 2311004 Guizhou, Pearl River PP112595 This study Sinocyclocheilus punctatus 03 ZJFR 2312002 Guangxi, Pearl River PP112596 This study Sinocyclocheilus punctatus 04 GZNU 20150811002 Guizhou, Pearl River MK610341 NCBI Sinocyclocheilus longibarbus XH2901 Guizhou, Pearl River AY854714 NCBI Sinocyclocheilus longicornus GZNU 20210503016 Guizhou, Pearl River MZ634123 NCBI Sinocyclocheilus zhenfengensis GZNU 20150112021 Guizhou, Pearl River MK610342 NCBI Sinocyclocheilus bicornutus XH8301 Guizhou, Pearl River AY854730 NCBI Sinocyclocheilus angularis GZNU 202001332 Guizhou, Pearl River MW362289 NCBI Sinocyclocheilus xingyiensis GZNU SLS202008180 Guizhou, Pearl River ON573221 NCBI Sinocyclocheilus guanyangensis n/a Guangxi, Pearl River 0Q718399 NCBI Sinocyclocheilus lateristriatus XH1601 Yunnan, Pearl River AY854707 NCBI Sinocyclocheilus malacopterus XHO901 Yunnan, Pearl River AY854697 NCBI Sinocyclocheilus angustiporus XH1203 Yunnan, Pearl River AY854702 NCBI Sinocyclocheilus hyalinus XH4701 Yunnan, Pearl River AY854721 NCBI Sinocyclocheilus rhinocerous XH3901 Yunnan, Pearl River AY854720 NCBI Sinocyclocheilus tingi YNUST 201406180002 Yunnan, Pearl River MG323567 NCBI Sinocyclocheilus guishanensis XH5401 Yunnan, Pearl River AY854722 NCBI Sinocyclocheilus maculatus n/a Yunnan, Pearl River MF325010 NCBI Sinocyclocheilus maitianheensis XH2301 Yunnan, Pearl River AY854710 NCBI Sinocyclocheilus anophthalmus XH3002 Yunnan, Pearl River AY854716 NCBI Sinocyclocheilus qiubeiensis n/a Yunnan, Pearl River MF324998 NCBI Sinocyclocheilus qujingensis XH3801 Yunnan, Pearl River AY854719 NCBI Sinocyclocheilus purpureus IHB 2006637 Yunnan, Pearl River EU366194 NCBI Sinocyclocheilus lunanensis XHO302 Yunnan, Pearl River AY854686 NCBI Sinocyclocheilus huaningensis XH3701 Yunnan, Pearl River AY854718 NCBI Sinocyclocheilus oxycephalus XHO201 Yunnan, Pearl River AY854685 NCBI Sinocyclocheilus yangzongensis XH6102 Yunnan, Pearl River AY854726 NCBI Sinocyclocheilus macrocephalus XHO110 Yunnan, Pearl River AY854684 NCBI Sinocyclocheilus yishanensis n/a Guangxi, Pearl River MK387704 NCBI Sinocyclocheilus macrophthalmus XH8401 Guangxi, Pearl River AY854733 NCBI Sinocyclocheilus xunlensis IHB 04050268 Guangxi, Pearl River EU366187 NCBI Sinocyclocheilus lingyunensis XHO502 Guangxi, Pearl River AY854691 NCBI Sinocyclocheilus donglanensis ASIZB 94746 Guangxi, Pearl River AB196440 NCBI Sinocyclocheilus ronganensis n/a Guangxi, Pearl River KX778473 NCBI Sinocyclocheilus macrolepis XH8201 Guangxi, Pearl River AY854729 NCBI Sinocyclocheilus anatirostris XH1901 Guangxi, Pearl River AY854708 NCBI Sinocyclocheilus anshuiensis n/a Guangxi, Pearl River KRO69120 NCBI Sinocyclocheilus microphthalmus XHO402 Guangxi, Pearl River AY854687 NCBI Sinocyclocheilus tianeensis XH3403 Guangxi, Pearl River AY854717 NCBI Sinocyclocheilus furcodorsalis XH2202 Guangxi, Pearl River AY854709 NCBI Sinocyclocheilus altishoulderus XH5801 Guangxi, Pearl River AY854724 NCBI Sinocyclocheilus jiuxuensis XH8501 Guangxi, Pearl River AY854736 NCBI Sinocyclocheilus jii XH8101 Guangxi, Pearl River AY854727 NCBI Sinocyclocheilus yimenensis IHB 2006645 Yunnan, Red River EU366192 NCBI Cyprinus carpio (outgroup) n/a n/a MK088487 NCBI zse.pensoft.net Zoosyst. Evol. 100 (2) 2024, 515-529 519 ets Ns 4 = vey fv a ~ —_—— Figure 2. Sinocyclocheilus guiyang, I]HB 202012250001, holotype, 124.0 mm SL; China: Guizhou Province: Gutyang City: Qin- gzhen County: Yangtze River Basin. A. Lateral view; B. Lateral, dorsal and ventral view of head; C. Micro-CT graph and recon- structed pharyngeal dentition; D. Live photo. Scale bar: 1 cm. zse.pensoft.net 520 following characters: tip of maxillary barbel not reaching to posterior edge of preoperculum, horn-like structure in forehead absent, eye absent or highly reduced, pectoral fin not significantly extending beyond base of pelvic fin. The major diagnostic characters for S. guiyang and relat- ed species are summarised in Table 3. Description. Morphometric measurements of type specimens have been transferred to percentage of stan- dard length (SL), as summarised in Table 2. Body lateral- ly compressed; maximum body depth positioned at inser- tion 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- Table 2. Morphometric characters of Sinocyclocheilus guiyang. Character Holotype Standard length (mm) 124.0 In Percentage of SL (%) Body depth 29.8 Predorsal length 55.4 Dorsal-fin base length 16.8 Dorsalfin length 18.8 Pre-anal length 71.4 Analfin base length 9.8 Analfin length 16.6 Prepectoral length 3153 Pectoral-fin base length 4.4 Pectoral-fin length 21.3 Prepelvic length 50.6 Pelvic-fin base length ae Pelvic-fin length 16.5 Caudal peduncle length (CPL) 19.5 Caudal peduncle depth (CPD) 11.8 Head length o1:5 Head depth 19.0 Head width 16.0 Snout length 10.9 Eye diameter 3:9 Interorbital width 10.4 Prenostril length Bat Width between posterior nostrils 7.2 Upper jaw length LOK Lower jaw length 9.4 Mouth width 8.3 Rostral barbel length BZ Maxillary barbel length 5.9 CPD to CPL ratio 0.60 zse.pensoft.net Shao, W.-H. et al.: A new troglobitic Sinocyclocheilus species from China 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-an- gulars not close at isthmus. Ten outer rakers (1) on first gill arch. Pharyngeal teeth pattern 1,3,44,3,0 (1); tooth tip pointed and compressed. Vertebrae 36 (2) (Fig. 2C). Dorsal fin with 3 unbranched and 8 (5) or 9 (2) branched rays, with last one divided at base; dorsal-fin length being 20.2% (17.6-24.5%) SL; origin closer to snout tip than to caudal-fin base; distal margin slightly concave, last unbranched ray strong, with serration on posterior edge; last unbranched ray split to base. Pectoral fin with 1 unbranched and 14 (6) or 15 (1) branched rays; tip extending to pelvic-fin insertion; pectoral-fin length being 21.5% (20.8-22.2%) of SL. Pelvic fin with 1 un- branched and 7 branched rays; inserted slightly posterior to dorsal-fin origin; tip not reaching to anus. Anal fin with 3 unbranched and 5 branched rays, last one divided at base; distal margin slightly concave; origin closer to pel- vic-fin insertion than to caudal-fin base. Caudal fin deeply forked, with 17 (6) or 18 (1) branched rays; upper and lower lobes pointed. Holotype + Paratypes (n = 7) Range Mean SD 57.5-144.1 26.7-33.1 29.4 2.1 53.6-59.6 55.9 2Al: 15.3-16.8 15.8 0.8 17.6-24.5 20.2 2:2 70.8-75.7 ae 1.8 7.5-10.2 9.2 1.0 16.0-18.3 LAD 0.9 30.7-35.7 325 1.6 4.0-4.6 4.3 0.2 20.8-22.2 21.5 0.5 50.6-55.3 22g 1,6 4.5-5.7 fv 0.4 15.4-19.8 17.3 1.4 16.5-20.7 Rete) 15 10.0-13.2 talk 2 1.2 30.2-34.1 32:2 1.4 16.8-21.0 19.14 13 10.4-17.6 1533 2.4 9.9-11.0 10.5 0.5 3.5-5.9 4.6 142 8.3-10.5 2 0.8 4.5-6.0 5.6 0.5 5.8-7.4 6.6 0.6 10.1-10.3 10.2 0.1 8.9-9.5 2 0.2 6.8-8.3 75 0.6 4,.9-7.1 6.2 0.9 5.8-8.3 TA 0.9 0.55-0.67 0.61 0.04 Zoosyst. Evol. 100 (2) 2024, 515-529 yell Table 3. Major diagnostic characters for Sinocyclocheilus guiyang and its close congeners. n/a, not available. ” ” = a) ” — wp 8 g 2 2 2 2 = 2 5 8 8 5 S 5 2 2 bo > =} rz) a 4 g 6 © 3 Characters Ss 2 © x * x = :Q N ap = 3 5 £ 2 % B = . 5 = = 4 E 7) a 7) 7) E s S 7) fs “” “” Eye Absent Normal Normal Absent Absent Normal Normal Normal Normal or highly reduced Tip of maxillary Reaching Reaching Reaching Notreaching Reaching Reaching or Extending Notreaching Extending barbel to posterior toposterior to anterior toanterior toposterior extending toanterior toanterior — to anterior edge of edge of edge of edge of edge of toposterior edge of edge of edge of orbit preoperculum orbit orbit orbit edge of orbit orbit orbit orbit Pectoral-fin Yes No Yes Yes Yes Yes Yes Yes No extending to pelvic-fin insertion Gill rakers 10 n/a 7-8 7 13-14 8-10 7-9 8-9 8-11 Lateralline 45-47 53-60 48-60 4l 38-41 42-51 47-50 49-51 71-81 scales Degenerated Yes Yes Yes Yes No Yes Yes Yes Yes body scales above and below lateral line Black blotches — Absent Present Present Absent Absent Absent Absent Absent Present on body Body covered with small scales, partially embedded subcutaneously; scales on lateral line slighter larger than other. Lateral line complete and horizontal, with 45 (4), 46 (2) or 47 (1) perforated scales. Scale rows above lat- eral line 20 (1), 21 (3), 22 (2) or 24 (1); below 13 (2) or 14 (5). Circumpeduncular scales 32 (1), 33 (1), 34 (2), 35 (2) or 36 (1). 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 dor- sal-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 Wuyjiang River in the upper Yangtze River Basin in Qin- gzhen County, Guiyang City, Guizhou Province, China (Fig. 4). The species inhabits pools of subterranean stream with gravel substrate (Fig. 5). Video record of Sinocy- clocheilus 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 ‘pr hH 422% 4G)’ (Guiyang Golden-line Barbel). Morphometric comparisons. Principal component analysis for Sinocyclocheilus guiyang, S. punctatus, S. multipunctatus and S. sanxiaensis, based on 29 log-trans- formed 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, re- spectively. In the PC1 vs. PC3 scatter plot, S. guivang 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 poste- rior 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 1s sep- arated from S. punctatus on the PC2 axis in the PC1 vs. PC2 scatter plot (Fig. 6B). Eye diameter, maxillary bar- bel 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. puncta- tus by shorter maxillary (7.2-13.3% SL vs. 5.8-8.3% in S. guiyang, 4.9-9.4% in S. punctatus) and rostral barbel lengths (6.0-10.7% SL vs. 4.9-7.1% in S. guiyang, 4.9- zse.pensoft.net 522 Shao, W.-H. et al.: A new troglobitic Sinocyclocheilus species from China 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 mid- line from the nape to the dorsal-fin origin. 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 in- cluded in the aligned dataset of cytb gene, with 661 conser- vative sites, 473 variable sites, 390 parsimony informative zse.pensoft.net 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 lin- age 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- Zoosyst. Evol. 100 (2) 2024, 515-529 107°0O'E 27°0'N 26°0'N Figure 5. Habitat of Sinocyclocheilus guiyang. A. The pool of a subterranean stream where S. guivang was collected; B. S. gui- yang in situ. B23 108°O'E » | Legend as guiyang HS. multipunctatus : © S. cyphotergous 24 \_~\ Province boundary Drainage divide \A\N Yangtze River system S : \AYN Pearl River system Elevation(m) mame 23000 —_ , 0 20 40 80 km ee cies clustered with the lineage comprising sequences of S. multipunctatus, S. cyphotergous and S. sanxiaensis. Addi- tionally, the topology of phylogenetic reconstruction in the present study supports the monophyly of the S. cyphoter- gous — S. multipunctatus species group. Average genetic distances derived from cytb sequences of Sinocyclochei- /us 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 struc- ture and degenerated (lost or highly reduced) eye. It is dis- tinct 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%). zse.pensoft.net 524 Shao, W.-H. et al.: A new troglobitic Sinocyclocheilus species from China Table 4. PCA loadings of the first three principal components extracted from 29 morphometric data for Sinocyclocheilus guiyang and related species. S. guiyang, S. punctatus, S. guiyang, S. punctatus S. multipunctatus, S. sanxiaensis PC1 PC2 PC3 PC1 PC2 PC3 Standard length 0.991 -0.062 0.028 0.995 0.026 0.045 Body depth 0.948 -0.034 -0.165 0.966 0.049 0.204 Predorsal length 0.991 0.035 0.022 0.996 0.008 0.050 Dorsalfin base length 0.978 -0.090 0.030 0.989 0.011 0.027 Dorsalfin length 0.928 0.027 0.019 0.895 0.115 0.376 Pre-anal length 0.986 -0.058 0.037 0.995 0.020 0.015 Analfin base length 0.967 0.107 -0.041 0.974 0.020 -0.104 Analfin length 0.962 0.032 0.075 0.985 0.073 0.101 Prepectoral length 0.990 0.029 -0.071 0.992 -0.024 0.074 Pectoral-fin base length 0.712 0.655 0.193 0.963 0.161 -0.080 Pectoral-fin length 0.978 0.011 -0.043 0.970 -0.092 0.132 Prepelvic length 0.991 -0.040 0.020 0.996 -0.005 -0.008 Pelvic-fin base length 0.732 0.636 0.097 0.954 0.019 0.228 Pelvic-fin length 0.976 0.002 0.134 0.987 0.097 0.056 Caudal peduncle length 0.954 -0.074 0.049 0.958 0.092 -0.170 Caudal peduncle depth 0.957 -0.019 -0.065 0.956 -0.107 -0.230 Head length 0.990 0.035 0.072 0.997 0.008 0.034 Head depth 0.991 -0.053 -0.046 0.996 -0.014 0.000 Head width 0.972 -0.080 0.048 0.970 -0.104 0.006 Snout length 0.966 0.183 0.094 0.983 -0.056 0.029 Eye diameter 0.716 0.389 0.408 0.758 0.632 0.056 Interorbital width 0.982 0.010 -0.086 0.979 0.147 0.107 Prenostril length 0.961 0.052 -0.036 0.954 0.150 0.045 Width between posterior nostrils 0.956 0.009 0.199 0.959 -0.183 -0.170 Upper jaw length 0.962 -0.116 0.179 0.983 -0.040 0.108 Lower jaw length 0.964 -0.118 -0.156 0.986 -0.008 0.103 Mouth width 0.958 0.031 0.052 0.954 0.155 -0.003 Rostral barbel length 0.731 -0.440 0.492 0.956 -0.161 0.081 Maxillary barbel length 0.764 -0.300 0.524 0.938 0.185 0.169 Table 5. Average uncorrected pairwise genetic distance (p-distance, %) derived from cytb in 15 species of Sinocyclocheilus dis- tributed in Guizhou Province or the Yangtze River Basin. Bold numbers, intraspecific distances; regular numbers, interspecific distances; n/a, not available. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1. S. guiyang 0.1 2. S. multipunctatus 2.5 0.4 3. S. punctatus 23 “26 O07 4. S. cyphotergous 2e bil, Sil 10:8 5. S. sanxiaensis 3:2 glv@ =" 29. “el6 inva 6. S. longibarbus 10.92 0.1.0, “129 Tie ley nika 7. S. grahami ee Tee rebhss 9 eee eho nse 8. S. wumengshanensis 12.3. 11.8 12.1 12.1 12.3 11.7 42 n/a 9. S. angustiporus Pleas LE 21S Dhoes loads 7 tlh -nfa 10. S. macrolepis Ss Did Los Die TOF hi ley 126-118: ~~ nxa 11. S. bicornutus 10.8 10.9 11.4 11.1 11.4 10.3 12.2 124 11.1 #4113 n/a 12. S. zhenfengensis TOs VLO8" dle TOS TLS LOO ize beAe I3 Li 929". nva 13. S. angularis V2) 12" PRT PLS se 39.6 L823 2 ee, 28 224 insa 14. S. longicornus 19 ALIS EIS aS Tea hOB Pars 2 ee ss. Tine bso eb: -Stf. enxa 15. S. xingyiensis TAR ALT is rn TS TOs! Tee tes. dikes) “120” 2isi i299