Zoosyst. Evol. 98 (1) 2022, 93-107 | DOI 10.3897/zse.98.80547 > PENSUFT. Ate BERLIN Gobiobotia lii, a new species of gudgeon (Teleostei, Gobionidae) from the middle Chang-Jiang Basin, central China, with notes on the validity of G. nicholsi Banarescu & Nalbant, 1966 Xiao Chen!*, Man Wang", Liang Cao!, E Zhang! 1 The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China 2 University of Chinese Academy of Sciences, Beijing, China http://zoobank.org/00AEDECC-7652-42F6-9DC9-5F4D77FEI8BD Corresponding author: E Zhang (zhange@ihb.ac.cn) Academic editor: Nicolas Hubert # Received 17 January 2022 # Accepted 11 March 2022 Published 29 March 2022 Abstract Gobiobotia lii is described from the Qi-Shui, a stream tributary on the northern bank of the middle Chang-Jiang mainstem in Hubei Province and Lake Dongting in Hunan Province, central China. The new species is distinguished from all other congeneric species by possessing a combination of the following characters: a naked region of the abdomen adjacent to the ventral mid-line extending to the vent and the vertebral count (4+3 1-32). The validity of G. /ii is confirmed by its monophyletic nature recovered in a phylogenetic analysis, based on the cyt b gene and its significant sequence divergence with sampled congeneric species. Critical notes were given on the species recognition of historically documented eight-barbel gudgeons co-existing in Lake Dongting. Gobiobotia nicholsi Banarescu & Nalbant, 1966 should be a valid species distinct from G. filifer (Garman, 1912) and both G. pappenheimi Kreyenberg, 1911 and G. boulengeri (=Xenophysogobio boulengeri (Tchang, 1929)) have an erroneous record from the Lake. Key Words cyt b gene, new taxon, morphology, species identification, taxonomy Introduction The gudgeon genus Gobiobotia Kreyenberg, 1911 (type species G. pappenheimi) comprises about 19 species (Fricke et al. 2021), characterised by the combination of four pairs of barbels; a gas bladder with a transversely widened or oval encapsulated anterior chamber and a minute free posterior chamber, but without pneumatic duct; and large scales, with five to six scale rows above the lateral line (He and Chen 1998). They are typically small-sized bottom dwellers hitherto recorded from the major river basins of the Korean Peninsula, Taiwan and Hainan Island, continental China and northern Vietnam (He and Chen 1998; Kottelat 2001a; Kim and Park 2002). The taxonomic history of Gobiobotia has been uneven, with the enigmatic genus often placed in a separate sub- family or family of its own (Kottelat 2001a). Species of this genus and Xenophysogobio Chen & Cao, 1977, an endemic Chinese genus currently known from the upper Yangtze River (= Chang-Jiang in Chinese) Basin, were referred to the subfamily Gobiobotinae of the Cyprinidae (He and Chen 1998). All eight-barbel gudgeons were re- cently referred to the family Gobionidae (Tan and Arm- bruster 2018). Nonetheless, the monophyletic nature of the either Gobionidae or Gobiobotia has been confirmed in many molecular phylogenetic studies of the order Cypriniformes (Yang et al. 2006; Tang et al. 2012; Zhao et al. 2016; Li et al. 2018). Gobiobotia, especially from China, has a taxonomic in- ertia despite He and Chen’s (1998) taxonomic revision of the genus. For example, the eight-barbel gudgeons from the mid-lower Chang-Jiang Basin are traditionally classified as four species: G. brevirostris Chen & Cao, 1977, G. fil- ifer (Garman, 1912), G. meridionalis Chen & Cao, 1977 Copyright Chen, X. 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. 94 and G. tungi Fang, 1933 (Zhang and Zhao 2016). Howev- er, G. jiangxiensis, originally described by Zhang and Liu (1995) from the Poyang Lake Basin, was not included in Gobiobotia by He and Chen (1998). Gobiobotia nicholsi, initially described by Banarescu and Nalbant (1966) from Lake Dongting, was regarded as a synonym of G. ichan- gensis Fang, 1930 (Chen and Cao 1977), a species subse- quently synonymised with G. filifer (Ding 1994). Although this synonym is widely accepted by succeeding Chinese workers, the validity of G. nicholsi requires re-evaluation, based on examination on its type specimens. Fish surveys were conducted during 2011—2015 and 2017-2018 in Lake Dongting in Hunan Province and during 2021 in some tributaries on the northern bank of the middle Chang-Jiang in Hubei Province, central Chi- na. These surveys yielded eighty-five specimens referred to as Gobiobotia, conforming to G. filifer and G. meridi- onalis, as well as a morphologically and genetically dis- tinct subsample which represents an undescribed species. The present study aims to provide a description of this unnamed species. Some notes on the validity of G. nich- olsi and the identification of other historically document- ed co-existing eight-barbel gudgeons in Lake Dongting are also provided. Material and methods Specimen sampling and preservation. Specimens utilised for this study were sampled in accor- dance with the Chinese Laboratory Animal Welfare and Ethics animal welfare laws (GB/T 35892-2018). After being anaesthetised, all captured individuals were fixed by immersion in ethanol or formalin. Specimens were collected using gill nets, trap nets and electrofishing. Caught specimens of Gobiobotia were stored in 10% formalin for morphological examination or 95% ethyl alcohol for DNA extraction. All sequences amplified in this study were submitted to GenBank. Their voucher specimens are deposited in the collection of the Museum of Aquatic Organisms at the Institute of Hydrobiology (THB), Chinese Academy of Sciences (CAS). Morphological analysis Measurements were taken point to point with a digital cal- iper connected directly to a data-recording computer and data recorded to the nearest 0.1 mm. Measurements were taken on the left side of specimens whenever possible, following methods used by Kottelat (2001b) and Song et al. (2018). The head length and measurements of other parts of the body are given as percentages of standard length (SL). Measurements of parts of the head are giv- en as proportions of the head length (HL) (Tables 1, 2). The counts of vertebrae were taken from radiographs of Micro-CT or X-rays. The specimens examined in this study are deposited in the collections of: zse.pensoft.net Chen, X. et al.: Gobiobotia Iii and G. nicholsi AMNH American Museum of Natural History, New York; BMNH _ Natural History Museum, London; THB Institute of Hydrobiology, Wuhan; MNHN~ Muséum National d’ Histoire Naturelle, Paris; ZMB Museum fiir Naturkunde-Leibniz Institute for Evolution and Biodiversity Science, Berlin. DNA extraction, amplification and sequencing Genomic DNA was extracted from fin clips stored in ethanol using the TIANamp Genomic DNA Kit (Tian- gen Biotech, Beijing) with the recommended protocol.. The cyt b gene was amplified by using primers L 14724 (GACTTGAAAAACCACCGTTG) and H15915 (CTC- CGATCTCCGGATTACAAGAC) adopted from Xiao et al. (2001). The mitochondrial gene was performed in 25 ul volumes with 12.5 ul Master mix Taq (Beijing TsingKe Biotech Co., Ltd.), 1 wl of each primer, 1 ul template DNA, adding double distilled water (dd H,O) to supply the volume. The thermocycling conditions were as follows: initial denaturation for 4 min at 94 °C, de- naturation for 50 s at 94 °C, annealing for 50 s at 55 °C and extension for 1 min, in terms of the product length at 72 °C. After 34-35 cycles, the final extension was done at 72 °C for 10 min; the product was then stored at 4 °C. The sequencing was finished by Tianyihutyuan Biotech- nology Company. Phylogenetic analyses The cyt 6 gene of around 1100 bp base was chosen for phylogenetic analysis. The amplified 48 gene sequenc- es were utilised for molecular phylogenetic analysis along with another 11 GenBank-retrieved sequences of the same gene from six congeneric species and two out- groups (Microphysogobio xianyouensis and M. fukiensis). Detailed information about samples, used here for mo- lecular analysis, is given in Table 3. The sequences were aligned using MAFFT 7.0 (Katoh and Standley 2013) and ends trimmed. The genetic distance was calculated with MEGA 7.0, based on the uncorrected p-distance model (Kumar et al. 2016). PhyloSuite (Zhang et al. 2020) was used for phyloge- netic analyses. The selection of the best-fit model of nucle- otide evolution, based on Akaike’s Information Criterion was performed in ModelFinder (Kalyaanamoorthy et al. 2017). MrBayes 3.2.6 (Ronquist et al. 2012) was utilised for Bayesian analysis with the selected model: GTR+I+G+F, applying the optimal nucleotide evolution model and the MCMC method with four chains (three hot chains and one cold chain) running simultaneously for 20,000,000 genera- tions to calculate posterior probability. Trees were sampled for every 1000 cycles. The initial 25% of sampled data were discarded as burn-in. Sufficient mixing of the chains was considered to be reached when the average standard deviation of split frequencies was below 0.01. Zoosyst. Evol. 98 (1) 2022, 93-107 95 Table 1. Morphometry of Gobiobotia lii. Measurements are in percentage of standard length, except for standard length (SL). SD, standard deviation; r, Pearson’s correlation coefficient; p, significance; linear regression parameters calculated from measurements. HT = Holotype. Measurements N HT Min Max Mean SD slope (b) intercept (a) r (SL) p SL (mm) 14 A38.6 “42.1. 51:2 A468 2./ Body depth 14 22.6 156 22.3 19.6 2 0.5461 5.7924 0.701 0.005 Head length 14 252le 2225" “25 “23:5 0.8 0.0124 23.043 0.037 0.9 Pre-dorsal length 14 AS 5» 447. bleet pare: Lid -0.029 48.906 -0.04 0.883 Pre-pectoral length 14 Ale 204° 42332) ae ® 0.9 -0.154 29.126 -0.47 0.091 Pre-pelvic length 14 AS: “aa 620° - ABs) 1.9 0.1722 56:232 -0.25 0.398 Pre-anal length 14 74.8 70.4 78.4 74 2.4 -0.0537 76.607 -0.06 0.837 Dorsal length 14 252 “2101 26 24 1.6 0.3178 9.2449 0.541 0.046 Pectoral length 14 2228.- WUSele <23.4- gid 1.6 -0.1957 30.756 -0.34 0.24 Pelvic length 14 196 15.9 19.6 18 1.2 -0.1486 24.994 -0.33 0.257 Anal length 14 20:1. lei2’ -204 © 2384 1.4 -0.0407 20.437 -0.08 0.795 Pectoral-pelvic distance 14 29.2 26 30:3: 370.4 PRG) -0.0534 30.95 -0.11 02705 Pelvic-anal distance 14 27.2 249 30 27,5 15 0.2996 41.551 -0.54 0.046 Pelvic-anus distance 14 13.2 9.6 ieaiven T16 ba 0.0514 14.082 0.12 0.682 Anus-anal distance 14 164.- 81237 184. 54 1.7 0.1019 10.664 0.164 0.575 Head depth 14 61.6 52 69s S615 4.9 0.7576 39.821 0.54 0.046 Head width 14 73.1 65.4 80.8 75.4 3.8 -0.066 64.639 -0.04 0.901 Snout length 14 40.1 389 43.2 41.8 1.1 0.007 41.347 0.017 0.955 Interorbital width 14 20:8 20H © 28:6 -246 2 0.0054 24.079 0.007 0.982 Eye diameter 14 19.1 20 25,0 «22:5 1.7 0.2117 32.172 -0.3 0.29 Maxillary barbel length 14 20:/ 23:9 67:9 33 10.5 0.6906 0.2997 0.18 0.539 Post-orbital length 14 Bo-o= S125: 46:6, 395 5 0.355 bb 27 0.19 0.526 Table 2. Morphometric measurements for four species of Gobiobotia: G. Jiangxiensis, G. pappenheimi, G. meridionalis and G. filifer. Character G. jiangxiensis (n=6) G. pappenheimi(n=14) G. meridionalis (n = 19) G. filifer (n = 21) Range Mean+SD Range Mean+SD Range Mean+SD Range Mean+SD SL (mm) 35.2-52.5 40.245.8 27.9-56.1 43.74+8.7 54.0-99.5 80.4+10.8 52.2-96.5 75.5+11.7 Morphometric data % of SL Body depth 12.2-22.5 17.143.3 27.9-56.1 43.7+8.7 14.9-20.8 17.941.5 15.4-22.5 18.2+1.9 Head length 22.0-25.9 24.44+1.3 12.5-14.5 13.640.7 24.4-28.1 26.741.0 17.9-28.2 25.7+2.1 Pre-dorsal length 46.8-50.5 48.641.6 46.2-51.8 49.2+1.6 43.8-51.9 46.542.0 41.2-52.7 46.8+2.4 Pre-pectoral length 19.8-24.1 22.941.6 18.8-28.1 23.642.8 24.6-27.3 26.040.9 23.2-28.5 25.6+1.3 Pre-pelvic length 44.6-50.8 48.342.3 44.7-50.7 47.7+1.9 45.0-52.4 48.341.7 43.5-51.1 47.8+2.0 Pre-anal length 71.0-78.1 74.74+2.2 69.4-79.0 75.242.4 72.7-77.5 75.041.4 69.0-77.3 73.3+2.1 Dorsal length 27.1-31.2 28.841.6 22.8-28.1 24.741.5 23.4-27.5 25.641.1 19.8-25.6 23.1+1.6 Pectoral length 25.1-28.2 26.541.0 17.0-22.5 20.141.6 21.9-29.1 25.041.5 22.9-31.8 26.1+2.1 Pelvic length 18.1-23.4 20.641.7 16.0-21.4 19.2+1.4 18.0-22.1 20.541.2 15.2-20.0 17.8+1.2 Anal length 18.1-21.3 19.141.0 16.0-21.4 19.2+1.4 17.5-20.9 19.0+0.9 15.5-19.2 17.0+0.9 Pectoralpelvic distance 26.3-28.7 27.340.8 22.9-27.1 25.141.4 21.0-28.1 24.241.7) 21.9-26.0 24.1+1.1 Pelvic-anal distance 24.4-26.1 25.540.6 25.7-31.9 28.141.9 26.9-31.1 28.541.2 23.9-29.9 26.2+1.6 Pelvic-anus distance 11.7-14.9 12.9+1.1 8.2-15.4 11.0+#1.7 9.0-14.5 11.8+1.2 7.6-13.1 10.8+#1.3 Anus-anal distance 11.3-14.3 12.740.9 14.0-18.1 16.041.2 14.2-19.1 16841.4 144-199 16.4+1.6 % of HL Head depth 54.4-60.3 57.141.9 47.5-59.0 53.643.7 51.3-59.0 55.442.2 47.1-76.1 53.9+6.1 Head width 60.9-82.1 70.446.2 51.3-73.2 67.4+5.3 55.4-70.9 63.024.0 49.8-79.4 59.4+7.2 Snout length 40.5-46.4 42.642.0 33.7-41.8 38.042.0 41.6-62.7 46.024.8 34.7-55.1 40.2+4.2 Interorbital width 21.5-29.3 24.8+2.6 17.0-29.1 21.8+3.6 16.6-29.1 21.043.8 24.9-40.7 28.843.2 Eye diameter 19.3-25.2 22.542.0 16.1-27.4 21.343.7) 22.1-35.8 26.043.7. 12.1-23.2 16.1+2.6 Maxillary barbel length 15.4-19.0 16.941.3 20.1-45.4 31.6469 20.3-39.5 35.24+2.7 25.6-37.4 31.1+43.8 Post-orbital length 35.7-38.2 37.141.0 33.5-44.4 39.142.9 32.1-50.4 37.2+4.7 42.0-60.5 46.7+3.7 Meristic counts Lateral-line scales 38-39 38.4+0.5 37-39 38.1+0.7 41-43 41.8+0.6 40-42 40.8+0.7 Scale rows above 5-6 5.5+0.5 5-6 5.5+0.5 5-6 5.3+0.4 5-6 5.6+0.5 lateral line Pre-dorsal scales 12 1? 13 13 12-13 12.6+0.5 12-13 12.8+0.4 Circumpeduncular scales 12 12 12-13 12.4+0.5 Ps 12 12 1? zse.pensoft.net 96 Chen, X. et al.: Gobiobotia Iii and G. nicholsi Table 3. Detailed information on specimens used in this study. The species with * means the samples available in GenBank. Species Specimen voucher Sampling location GenBank no. G. lil IHB201711015435 Yuanjiang, Hunan Prov. (Lake Dongting, Chang-Jiang) OM275368 G. Iii IHB2017101929 Yuanjiang, Hunan Prov. (Lake Dongting, Chang-Jiang) OM275369 G. lil IHB202103050300 Yuanjiang, Hunan Prov. (Lake Dongting, Chang-Jiang) OM275370 G. lil IHB202103050303 Yuanjiang, Hunan Prov. (Lake Dongting, Chang-Jiang) OM275371 G. lil IHB201801016013 Yuanjiang, Hunan Prov. (Lake Dongting, Chang-Jiang) OM275372 G. lii IHB201 7097256 Qichun, Hubei Prov. (QiShui, Chang-Jiang) OM275373 G. lil IHB201 7097257 Qichun, Hubei Prov. (QiShui, Chang-Jiang) OM275374 G. lii IHB201 7097258 Qichun, Hubei Prov. (QiShui, Chang-Jiang) OM275375 G. lil IHB202103050858 Qichun, Hubei Prov. (QiShui, Chang-Jiang) OM275376 G. lil IHB202103050859 Qichun, Hubei Prov. (QiShui, Chang-Jiang) OM275377 G. filifer IHB201711015758 Maocaojie, Hunan Prov. (Lake Dongting, Chang-Jiang) OM275378 G. guilingensis IHB20161062A Rongshui, Guangxi Prov. (Zhu-Jiang) OM275379 G. tungi IHB202108056037 Shangrao, Jiangxi Prov. (Chang-Jiang) OM275380 G. tungi IHB202108056038 Shangrao, Jiangxi Prov. (Chang-Jiang) OM275381 G. meridionalis IHB201 7040446 Hengdong, Hunan Prov. (Xiang-Jiang, Chang-Jiang) OM275382 G. meridionalis IHB201 7040447 Hengdong, Hunan Prov. (Xiang-Jiang, Chang-Jiang) OM275383 G. meridionalis IHB2016106403 Chenxi, Hunan Prov. (Yuan-Jiang, Chang-Jiang) OM275384 G. meridionalis IHB2016106404 Chenxi, Hunan Prov. (Yuan-Jiang, Chang-Jiang) OM275385 G. meridionalis IHB201 7104358 Kaili, Guizhou Prov. (Yuan-Jiang, Chang-Jiang) OM275386 G. meridionalis IHB201 7104362 Kaili, Guizhou Prov. (Yuan-Jiang, Chang-Jiang) OM275387 G. meridionalis IHB201 7040817 Nanxiong, Guangdong Prov. (Zhu-Jiang) OM275388 G. meridionalis IHB201 7040832 Shixing, Guangdong Prov. (Zhu-Jiang) OM275389 G. meridionalis IHB201 7013891 Suichuan, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275390 G. meridionalis IHB201 7013893 Suichuan, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275391 G. meridionalis IHB201804014151 Fuzhou, Jiangxi Prov. (Fu-He, Chang-Jiang) OM275392 G. meridionalis IHB201804014152 Fuzhou, Jiangxi Prov. (Fu-He, Chang-Jiang) OM275393 G. meridionalis IHB201 707014638 Ningdu, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275394 G. meridionalis IHB201 707012675 Ningdu, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275395 G. meridionalis IHB201 707018995 Taihe, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275396 G. meridionalis IHB201 7075488 Longnan, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275397 G. meridionalis IHB201 707017921 Shicheng, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275398 G. meridionalis IHB201707017918 Shicheng, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275399 G. meridionalis IHB201 707013207 Huichang, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275400 G. meridionalis IHB201707012757 Pingshan, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275401 G. meridionalis IHB2016104697 Jian, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275402 G. meridionalis IHB201 707018980 Taihe, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275403 G. meridionalis IHB201 707018994 Taihe, Jiangxi Prov. (Gan-Jiang, Chang-Jiang) OM275404 G. pappenheimi* Uncatalogued Tieling, Liaoning Prov. (Liao-He) NC032293.1 G. naktongensis* Uncatalogued Korean Peninsula JX104484.1 G. naktongensis* Uncatalogued Korean Peninsula JX104485.1 G. macrocephala* Uncatalogued Korean Peninsula JX046845.1 G. macrocephala* Uncatalogued Korean Peninsula JX046846.1 G. brevibarba* Uncatalogued Korean Peninsula JX094047.1 G. brevibarba* Uncatalogued Korean Peninsula JX094048.1 Xenophysogobio boulengeri* Uncatalogued Upper Chang-Jiang Basin AF375868.1 Xenophysogobio nudicorpa* Uncatalogued Upper Chang-Jiang Basin NC_025300.1 Microphysogobio xianyouensis* NTOUP 201011-534 Mulan-Xi, Fujian Prov. KM999931.1 Microphysogobio fukiensis* NTOUP 2015-10-001 Shaowu City, Fujian Prov. KT877353.1 Results 115°43'43"E; D. M. Guo, X. Gong and Y. Liu; 5 March 20210 Gobiobotia lii sp. nov. Paratypes. [HB 202103051399-1400, 202103050858- http://zoobank.org/0061399E-1D8E-4764-A475-9FFC29A 6490F 0859, 4 specimens, 45.1-48.0 mm SL, other data same Figs 1, 2 as holotype. THB 2014070560439-0447, 9 specimens, 42.1—51.2 mm SL. P. R. China: Hunan Province: Hanshou Gobiobotia pappenheimi Chen & Cao, 1977: 556 (Lake Dongting), City: Potou Town: Chang-Jiang Basin, Lake Dongting, Synonym 29°00'05"N, 111°58'31"E; L. Cao, Z. G. Jiang, S. J. Ren, R. X. Xie and X. Wang; 5 July 2014. IHB 2017101929, Holotype. [HB 202103051401, 48.6 mm SL. PR. 201711015435, 201801015997; 6012-6015; 6143-6146, China: Hubei Province: Qichun County: Xiangqiao 11 specimens, 35.0-40.1 mm SL. P. R. China: Hunan Town: Chang-Jiang Basin, Qi-Shui stream, 30°20'31"N, Province: Yuanjiang City: Liaodaokou: Chang-Jiang zse.pensoft.net Zoosyst. Evol. 98 (1) 2022, 93-107 97 Figure 1. Gobiobotia lii, holotype, IHB 202103051401, 48.6 mm SL, photographed alive immediately upon capture. P. R. China: Hubei Province: Qichun County: Xiangqiao Town: Chang-Jiang Basin, Qi-Shui. Figure 2. Dorsal (a), lateral (b) and ventral (c) views of Gobiobotia /ii, holotype, IHB 202103051401, 48.6 mm SL. P. R. China: Hubei Province: Qichun County: Xiangqiao Town: Chang-Jiang Basin, Qi-Shui. zse.pensoft.net 98 Basin, Lake Dongting, 28°51'39.02"N, 112°33'37.87"E; X. Chen, C. T. An, W. H. Shao and Z. T. Wang; 19 October 2017 and 1 January 2018. Diagnosis. Gobiobotia lii is distinct from all other congeneric species, except G. brevirostris Chen & Cao, 1977, G. homalopteroidea Rendahl, 1933, G. jiangxien- sis Zhang & Liu, 1995 and G. pappenheimi Kreyenberg, 1911, in having a naked region of the abdomen adjacent to the ventral mid-line extending to or beyond the vent (vs. to or away from the pelvic-fin base) (Figs 2c, 3). It differs from these four species in having 4+31-—32 (vs. 4+33—37) vertebrae. The new species shares with G. hom- alopteroidea and G. pappenheimi the presence of smaller Chen, X. et al.: Gobiobotia Iii and G. nicholsi eyes (diameter less than the interorbital width), maxillary barbels longer than the eye diameter and the third pair of longer mental barbels extending to the pectoral-fin inser- tion, these three characters separating them from G. brevi- rostris and G. jiangxiensis. The new species further differs from G. homalopteroidea in possessing a smaller (vs. larg- er) naked region of the abdomen adjacent to the ventral mid-line extending to the anus (vs. to the anal-fin origin) and the eye diameter 20.0—25.8% of HL (vs. 10.8—13.9%),; and from G. pappenheimi in having pectoral fins extending away from (vs. beyond) the pelvic-fin insertion, the second branched pectoral-fin ray not prolonged (vs. prolonged) and a longer (vs. shorter) snout than the post-orbital length. Figure 3. Ventral view of body in: (a) G. guilingensis IHB 202104053183, 86.7 mm SL. P. R. China: Guangxi Province: Rongshui County: Zhu-Jiang Basin, Rong-Jiang; and (b) G. meridionalis, [HB 201609021174, 84.8 mm SL. P. R. China: Hunan Province: Chenxi County: Chang-Jiang Basin: Yuan-Jiang. Description. Morphometric data for type specimens giv- en in Table 1. General body appearance of holotype shown in Figs 1 and 2. Body small and slender, with maximum depth at dorsal-fin origin. Pre-dorsal body profile depressed. Ventral profile slightly compressed. Abdomen flat or slight- ly convex from pectoral-fin insertion to anal-fin origin and slightly concave from anal-fin origin to caudal-fin base. Head depressed and broad. Snout pointed in lateral view and slightly bluntly rounded in dorsal view, longer than post-orbital head. Eyes small and dorsolateral; diame- ter less than interorbital width. Interorbital space flattened. Some irregular papillae present on the mental region. Mouth inferior, with opening laterally extending to the vertical line of front edge of nostril. Four pairs of barbels: one pair of maxillary barbels and three pairs of mental barbels. Maxillary barbels extending closely to a vertical line through middle of eye; first pair of mental barbels zse.pensoft.net short, inserted at the same level passing through maxil- lary-barbel roots and anterior to roots of second mental barbels and reaching anterior margin of eye; second pair of mental barbels extending beyond bases of third pair of mental barbels to hind margin of pre-opercula; third pair of mental barbels long, reaching pectoral-fin insertion. Fins rays flexible, dorsal fin with 3 simple and 7 (14 specimens examined) branched rays; pectoral fin with 1 simple and 12 (6) or 13 (8) branched rays, extending about two-thirds of the distance to pelvic-fin insertion. Pelvic fin with 1 simple and 7 (14) branched rays, reach- ing beyond the midway to anal-fin origin and surpassing anus; inserted closer to anal-fin origin than to anterior end of pectoral-fin base; located opposite to that of dor- sal-fin base. Anal fin with 3 simple and 6 (14) branched rays; origin equidistant from pelvic-fin insertion and caudal-fin base. Anus positioned closer to the pelvic-fin Zoosyst. Evol. 98 (1) 2022, 93-107 99 << KiKdkd, /// ] eal. alt Zul >1 pectoral and pelvic-fin insertions 9. Second branched pectoral-fin ray Not elongated Elongated Not elongated Not elongated Elongated 10. Vertebral counts 4431-32 (3) 4434 (2) 4+36-37 4+33-34 (6) 4+34-35 (5) 11. Lateralline pored scales 37-38 (14) 38-39 (2) 41-43 38-39 (6) 37-39 (14) ° From He and Chen (1998); “ from this study. included Misgurnus anguillicaudatus, Opsariichthys Sai acts at bidens and Parabotia fasciata. Etymology. The specific epithet is named after Shi- Zhen Li, a native of Qichun County where the holotype and partial paratypes were caught. Li was a well-known medical scientist in the Ming Dynasty, who compiled “Compendium of Materia Medica” (‘A424 A’ in Chi- nese) - one of the most valuable pieces of literature of traditional Chinese medicine. He had a typical image as an old man with a long white dense beard, just like the eight-barbel gudgeon. The common Chinese name ‘AS ER SAKE’ in here proposed for Gobiobotia Iii. Sequence variation and molecular phylogeny. A to- tal of 37 cyt b gene sequences from five Chinese species of Gobiobotia: G. filifer (one sequence), G. guilingen- sis (one), G. [ii (10), G. meridionalis (23) and G. tungi (two) were amplified in this study. These sequences were used for phylogenetic analysis along with another 11 GenBank-retrieved sequences from six congeneric spe- cies and two out-groups (Microphysogobio fukiensis and M. xianyouensis) (Table 3). A total length of 1038 bp gene sequence was obtained after sequence alignment and trim- ming, including 643 conserved sites, 396 variable sites, 349 parsimony informative sites and 47 singleton sites. The mean frequency of four nucleotides of the new species was A = 30.7%, T = 27.5%, C = 15.5% and G = 26.4%; the base composition was A-T rich (58.2%). The Bayes- ian Inference tree, based on the cyt b gene for G. /ii and other 10 congeners, is shown in Fig. 7, with node support values displayed. Intraspecific genetic distance values for here-recognised species of the Gobionidae ranged from 0.0% to 1.8%. The interspecific genetic distance of G. /ii with all other sampled congeneric species varied from 2.6% to 22.1% (mean 16.8%) and the intraspecific genet- ic distance value of this species was 0.8% (Table 5). In the Bayesian 50% mayority-rule consensus tree, samples of G. /ii constituted a robustly-supported (100% Bayesian posterior probability, bpp) lineage, strongly supported by 100% bpp to be sister of G. naktongensis Mori, 1935. Xenophysogobio nudicorpa Xenophysogobio boulengeri G. brevibarba G. macrocephala G. guilingensis 0.05 Figure 7. Bayesian Inference tree inferred from the cyt b gene for 11 putative species of eight-barbel gudgeons. Nodal num- bers are posterior probability values greater than 50%. Photographic examination on relevant Specimens Some of specimens, utilised in Banarescu & Nalbant’s (1966) taxonomic revision of Gobiobotia, are from Hup- ing (now Yueyang City) on Lake Tungting (= Dongting). These specimens were not examined by Chinese work- ers when they revised species of this genus. In this study, photographic examinations are made on them and also on the holotype of Xenophysogobio boulengeri and Gobio- botia pappenheimi and their topotypical specimens are examined as well. Both the holotype (AMNH 20523; Fig. 8) and the paratype (AMNH 20524) of G. nicholsi, as stated in its original description, have 44—45 lateral-line pored scales, four scale rows below the lateral line, the eye diameter about half of the interorbital width and the second branched pectoral-fin ray not prolonged. Nev- ertheless, the naked region of the abdomen adjacent to the ventral mid-line extends away from or to the pel- vic-fin insertion rather than to or behind the vent in the original description. Nineteen specimens (AMNH 10311) of 32.2-41.0 mm SL, under the name of G. pappenheimi from Lake Dongting, have a naked region of the abdomen adjacent to the ventral mid-line extending to the pelvic-fin insertion zse.pensoft.net 102 Chen, X. et al.: Gobiobotia Iii and G. nicholsi Table 5. Genetic distances (uncorrected p-distance) of cyt b gene computed by MEGA 11 amongst 11 species. Species Within Group 1 2 3 1. G. lii 0.0076 2. G. naktongensis 0.0078 0.0260 3. G. pappenheimi n/c 0.0387 0.0350 4. G. filifer n/c 0.1296 0.1212 0.1395 5. G. meridionalis 0.0029 0.6358." -0:1356-0.1352 6. G. guilingensis n/c 0.1721 0.1649 0.1824 7. G. tungi 0.0029 0.1736 0.1660 0.1757 8. G. brevibarba 0.0029 0.1826 0.1835 0.1853 9. G. macrocephala 0.0019 0.1856 0.1776 0.1814 10. X. boulengeri n/c 0.1833 0.1849 0.1800 11. X. nudicorpa n/c 0.1964 0.1822 0.1847 0.1996 4 5 6 7 8 a 10 0.1424 0.1575 0.1805 0.1856 0.1890 0.1925 0.1926 0.1864 0.2009 0.1927 0.1684 0.1740 0.1881 0.1993 0.1631 0.1787 0.1804 0.1979 0.1935 0.1914 0.1915 0.1883:..0.1912..°0.2209° 0.1199. 0;2070. 0.1199 Figure 8. Lateral (a) and ventral (b) views of G. nicholsi, AMNH 20523, holotype. and more than 40 lateral-line pored scales (Fig. 9a, b). By contrast, the holotype of G. pappenheimi (ZMB 18466) has a naked region of the abdomen adjacent to the ven- tral mid-line extending to the vent and 38—39 lateral-line pored scales (Fig. 9c, d), as found in specimens of this species from northern China. Two small specimens (AMNH 20522) of 30.0 mm and 31.2 mm SL, under the name of G. boulengeri (= Xenophysogobio boulengeri) from Lake Dongting, have larger eyes (diameter about one-fifth head length), the adpressed tip of the pectoral fin reaching or slight- ly beyond the pelvic-fin insertion, the adpressed tip of the pelvic fin reaching the anal-fin origin and the up- per extremity of the gill opening aligned with the up- per margin of the eye (Fig. 10a, b). Nevertheless, three available topotypical specimens and the holotype (MNHN-IC-1934-0180; photograph examined) have smaller eyes (diameter about one-tenth head length), the adpressed tip of the pectoral fin not reaching the pel- vic-fin insertion, the adpressed tip of the pelvic fin not reaching the anal-fin origin and the upper extremity of the gill opening aligned with the lower margin of the eye (Fig. 10c, d). zse.pensoft.net Discussion The generic concept of Gobiobotia still remains conten- tious. All eight-barbel gudgeons were traditionally clas- sified into three subgenera, namely Gobiobotia, Progo- biobotia and Xenophysogobio (Chen and Cao 1977). The generic rank was subsequently assigned to Xenophyso- gobio and Progobiobotia was rendered subgeneric to Gobiobotia (He and Chen 1998), a classification wide- ly accepted by succeeding authors, for example, Zhang and Zhao (2016) and Wu et al. (2021). The generic status of Gobiobotia and Xenophysogobio was verified in mo- lecular phylogenetic analyses of the gobiobotine fishes (Wang et al. 2002) or gudgeons (Tang et al. 2012; Li et al. 2018). However, the taxonomic status of the subgenus Progobiobotia (including two species G. (P.) abbreviata Fang & Wang, 1931, type species and G. (P.) guilingensis Chen, 1989) remained yet to be evaluated in these anal- yses as a result of failure to sample its included species. In the BI tree yielded from the cyt b gene (Fig. 7), G. (P.) guilingensis was distantly related to G. (G.) brevibarba Mori, 1935 and G. (G.) macrocephala Mori, 1935, but constituted a polytomy with G. (G.) tungi and a lineage Zoosyst. Evol. 98 (1) 2022, 93-107 103 Figure 9. Lateral (a) and ventral (b) views of G. pappenheimi, AMNH 10311, from Lake Dongting; lateral (c) and ventral (d) views of G. pappenheimi, ZMB 18466, holotype. consisting of G. (G.) filifer, G. (G.) Iii, G. (G.) meridio- nalis, G. (G.) naktongensis Mori, 1935 and G. (G.) pap- penheimi. Evidently, G. (P.) guilingensis was intertwined with sampled species of the subgenus Gobiobotia. For this reason, the subgeneric status of Progobiobotia 1s not warranted. The generic definition of Gobiobotia (sensu He and Chen 1998) is followed in this study. The pres- ence of four pairs of barbels can easily distinguish it and Xenophysogobio from all other genera of the Gobionidae. The genus Gobiobotia includes those eight-barbel gud- geons with a tiny free posterior chamber of the gas blad- der without pneumatic duct and large scales, with five or six scale rows above the lateral line. Species of the gudgeon genus Gobiobotia are subdi- vided into two groups, based on body squamation. One group includes those species with a naked region of the abdomen adjacent to the ventral mid-line, extending to the vent or even the anal-fin origin. Four species, along with G. /ii, are placed in the group: G. brevirostris, G. homalopteroidea, G. Jiangxiensis and G. pappenheimi. The rest of congeneric species are assigned to the other group defined by having an unscaled region of the abdo- men adjacent to the ventral mid-line extending to or away from the pelvic-fin base. There are marked variations of the new species with the most similar species, G. hom- alopteroidea and G. pappenheimi, which are provided in the diagnosis. Gobiobotia brevirostris is presently known only from the upper Han-Jiang of the middle Chang-Jiang Basin, while G. jiangxiensis occurs in the Xin-Jiang, an effluent of Lake Poyang. The new species is further dis- tinct from the two co-existing species in having 4+31—32 (vs. 4+33-—35) vertebrae, the smaller eyes (diameter less than the interorbital width), maxillary barbels longer than the eye diameter and the third pair of longer men- tal barbels extending to the pectoral-fin insertion; from G. brevirostris in having a longer (vs. shorter) snout than zse.pensoft.net 104 Chen, X. et al.: Gobiobotia Iii and G. nicholsi Figure 10. Lateral (a) and ventral (b) views of X. boulengeri, AMNH 20522, from Lake Dongting; lateral (c) and ventral (d) views of X. boulengeri, MNHN-IC-1934-0180, holotype. post-orbital head and relatively smaller (vs. larger) eye diameter than interorbital width; and from G. jiangxiensis in having longer (vs. shorter) maxillary barbels than the eye diameter and the third pair of mental barbels extend- ing to the pectoral-fin insertion (vs. to the front margin of the pre-opercula) (See Table 4 and Fig. 4). Some of type specimens of G. /ii were collected from Lake Dongting where five nominal species of eight-bar- bel gudgeons have been documented: G. boulengeri (= Xenophysogobio boulengeri), G. filifer, G. meridio- nalis, G. nicholsi and G. pappenheimi (Banarescu and Nalbant 1966; Chen and Cao 1977; He and Chen 1998: Wu et al. 2021). The identification of all these species, except the third one, indeed requires re-evaluation. From Chen and Cao’s (1977) point of view, type specimens of G. nicholsi were small and similar to G. ichangen- sis (= G. filifer) for the presence of small eyes, the first pair of mental barbels rooted anterior to maxillary-bar- bel bases and the well-developed keels on pre-dorsal scales of the dorsum; they also had no remarkable dif- ferences with small (25.5—32.0 mm SL) topotypes. These zse.pensoft.net characteristics led them to reach a conclusion that these type specimens were juveniles of G. filifer, a species cur- rently known from the Chang-Jiang Basin. Gobiobotia filifer is characterised by having an elongated tip of the second branched pectoral-fin ray (He and Chen 1998). This prolonged tip is definitely not exhibited by type specimens of G. nicholsi (AMNH 20523; Fig. 8). The observation by Gao et al. (1988) revealed that the second branched pectoral-fin ray of G. filifer has an elongated tip at the juvenile stage (16 mm TL). This finding ne- gates Chen and Cao’s (1977) hypothesis that the types of G. nicholsi were juveniles of G. filifer. Other characters diagnostic for G. filifer comprise 40—42 lateral-line pored scales, three scale rows below the lateral line and the eye diameter slightly less than the interorbital width (Chen and Cao 1977). They are not shared with the type speci- mens (photograph examined; Fig. 8) of G. nicholsi, with 44-45 lateral-line pored scales, four scale rows below the lateral line and the eye diameter about half of the inter- orbital width. It is apparent that G. nicholsi and G. filifer are two distinct species. Zoosyst. Evol. 98 (1) 2022, 93-107 Banarescu and Nalbant (1966) followed Nichols (1928; 1943) to identify specimens from Lake Dongting as G. pappenheimi, initially described by Kreyenberg (1911) from Tientsin (now Tianjin) of China, while giv- ing its precise type locality as the Pai-ho (= Bai-He of the Hai-He Basin) in Hopei (now Hebei Province). The orig- inal description is vague and, thus, of limited taxonomic use for current species identification. This species, as ex- hibited in the holotype (ZMB 18466, photograph exam- ined; Fig. 9c, d), has a naked region of the abdomen ad- jacent to the ventral mid-line up to the vent and a slightly prolonged tip of the second branched pectoral-fin ray and 38 lateral-line pored scales. These characters, however, are not shared with two large specimens (photograph ex- amined; Fig. 9a, b) recognised by Banarescu and Nalbant (1966) as G. pappenheimi from Lake Dongting. Instead, both have a naked region of the abodmen adjacent to the ventral mid-line up to the pelvic-fin base and more than 40 lateral-line scales and lacks a slightly elongated tip of the second branched pectoral-fin ray (Fig. 9a, b), thus not conspecific with either G. filifer or G. lii, as well as G. pappenheimi. Due to the two specimens and oth- er 17 small specimens (photograph examined) not pre- served in good condition, the precise count of lateral-line pored scales for them is impossible. All these specimens from Lake Dongting are probably the misidenfication of G. nicholsi. Nonetheless, the hypothesis needs to be con- firmed in a future study. In Chinese literature (Liang and Liu 1966; Chen and Cao 1977; He and Chen 1998; Zhang et al. 2016), the occurrence of G. pappenheimi in Lake Dongting is an erroneous record. This species is not found in the Chang-Jiang and river basin south of the river. One small specimen of 37.5 mm SL caught from Lake Dongting was referred to as a distinct species by Chen and Cao (1977), who stopped short of describing a new species owing to lack of large specimens. It has a naked region of the abdomen adjacent to the ventral mid-line up to the vent, no prolonged tip of the second branched pectoral-fin ray, the first pair of mental barbels inserted anterior to the maxillary-barbel bases, 38 lateral-line pored scales and a gas bladder with an anterior chamber enclosed in a membranous capsule (Chen and Cao 1977, page 556: table 10-1). All these characters, except the last one, are shared with G. /ii. It 1s possible that the small specimen from Lake Dongting is conspecific with this species. Size-related change is the plausible explanation for the variation in the anterior chamber of the gas blad- der between it and type specimens of G. /ii. Banarescu and Nalbant (1966) was the first to recog- nise two specimens from Lake Dongting of the middle Chang-Jiang Basin as G. boulengeri, a species that was initially described by Tchang (1929) from Szechwan (to- day’s Sichuan Province in the upper Chang-Jiang Basin). Both (AMNH 20522, photograph examined; Fig. 10a, b) have larger eyes (diameter about one-fifth head length), the adpressed tip of the pectoral fin reaching or slight- ly beyond the pelvic-fin insertion, the adpressed tip of 105 the pelvic fin reaching the anal-fin origin and the up- per extremity of the gill opening aligned with the upper margin of the eye. All these characters are incongruent with the original description of G. boulengeri (= Xeno- physogobio boulengeri). Three available topotypes and the holotype (MNHN-IC-1934-0180, photograph exam- ined; Fig. 10c, d) have small eyes (diameter about one of tenth head length), the adpressed tip of the pectoral fin not reaching the pelvic-fin insertion, the adpressed tip of the pelvic fin not reaching the anal-fin origin and the upper extremity of the gill opening aligned with the lower margin of the eye. Evidently, the aforementioned two specimens from Lake Dongting are not identical to X. boulengeri. To which species they belong still remains unclear, given the poor condition of specimens and no examination on the structure of their gas bladders. In Chinese literature (Chen and Cao 1977; He and Chen 1998; Zhang et al. 2016; Guo et al. 2021), X. boulengeri has an erroneous record from Lake Dongting. This spe- cies 1s endemic to the upper Chang-Jiang Basin. Based on the above analysis, it is here concluded that Lake Dongting harbours four eight-barbel gudgeons: G. filifer, G. Iii, G. meridionalis and G. nicholsi. The new Species is separated from all other three co-existing con- generic species by the presence of a naked region of the abdomen adjacent to the ventral mid-line extending to the vent (vs. extending away from or to the pelvic-fin base). It lacks an elongated tip of the second branched pecto- ral-fin ray typical for G. filifer and has 37-38 lateral-line pored scales fewer than 44—45 and 40-43 for G. nicholsi and G. meridionalis, respectively. The validity of G. /ii is further affirmed by its mono- phyletic nature recovered in cyt b gene-based phyloge- netic analysis and its significant sequence variation on all sampled congeners. In the BI trees (Fig. 7), Gobiobotia lii formed a strongly-supported exclusive lineage, being sister to the endemic South Korean species G. naktongen- sis. The new species differs from it in having, amongst others, a naked region of the abdomen adjacent to the ventral mid-line reaching the pelvic-fin base, the pecto- ral fin extending beyond the pelvic-fin insertion and the third pair of mental barbels reaching over the hind margin of the opercula. The sequence divergence of G. /ii with sampled congeneric species varied from 2.6—22.1% (av- erage 12.4%) (Table 5), greater than 2% being utilised as a threshold for vertebrates’ species delimitation (Avise and Walker 1999; Hebert et al. 2003). Comparative material G. abbreviata: 1HB2113-2117, 5 specimens, 53.4— 74.8 mm SL, Min-Jiang at Leshan City, Sichuan Prov- ince, China. G. brevibarba: THB, uncatalogued, 2 specimens, 61.5— 88.6 mm SL; Misan-Ri, Sangnam-Myon Inje-Gun, Kangwon-Do, South Korea. zse.pensoft.net 106 G. brevirostris. JHB 81VII1310-1311, 2 specimens, 39.0-41.7 mm SL; Han-Jiang at Tanghe County, Henan Province, China. G. filifer: THB 831V 1438-1439, 831V1441-1450, 64V2287, 641V0803-0804; THB 0305, 53120-53121, 53123, 20 specimens, 52.2-96.5 mm SL; Yichang City, Hubei Province, China. IHB 2017100792, 1 specimen, 78.6 mm SL; Yueyang City, Hunan Province, China. AMNH 79426, 1 specimen, all other data same as Yueyang City, Hunan Province, China (photograph examined). G. guilingensis: JHB 20161062A, 1 specimen, 86.7 mm SL; Zhu-Jiang at Rongshui County, Guangxi Province, China. G. jiangxiensis.: THB 90-IV-1256, holotype, 41.0 mm SL; Xin-Jiang at Shangrao City, Jiangxi Province. THB 90-IV-1257, THB 90-IV-1377, THB 90-IV-1731-1732, 4 paratypes, 37.0-54.0 mm SL; Shangrao City and Guangfeng County. IHB 90-IV-1796, 6 specimens, 35.2—52.5 mm SL; all other data same as holotype. G. macrocephala. THB, uncatalogued, 1 specimen, 53.8 mm SL; Somaegok-Ri pukpsng-Myon, Hongc- hon-Gun, Kangwon-Do, South Korea. G. meridionalis: THB 20160902 1340-1344, 201609056055, 6 specimens, 70.5-85.9 mm SL; Yuan-Jiang at Luxi County, Hunan Province, China. THB 201609025884- 5885, 2 specimens, 95.3-99.5 mm SL; Yuan-Jiang at Mayang County, Hunan Province, China. THB 201609056048, =201609021171-201609021176, 7 specimens, 75.2—93.7 mm SL; Yuan-Jiang at Chenxi County, Hunan Province, China. IHB 201711056049, 201809036686, 201809036690, 3 specimens, 54.0—74.3 mm SL; Xiang-Jiang at Chalin City, Hunan Province, China. G. naktongensis: IHB, uncatalogued, 2 specimens, 44.3— 47.5 mm SL; Kyongsangbuk-Do, South Korea. G. nicholsi: AMNH 20523, holotype, 35.7 mm SL; Yuey- ang City, Hunan Province, China (X-radiograph exam- ined); AMHN 20524, 2 paratypes, 21.9-32.2 mm SL; same locality as holotype (photograph examined). G. pappenheimi: ZMB 18466, holotype, 45.0 mm SL, Tianjin City, China (X-radiograph examined); BMNH 1925.8.6.36, Russia (X-radiograph examined); AMNH 10311, 10308, 10553, 19 specimens, 32.2-41.0 mm SL; Yueyang City, Hunan Province, China (photo- graph examined). [HB 80-VII-607-609, 611-613, 6 specimens, 45.8—56.1 mm SL; Nen-Jiang at Heilong- jiang Province, China. THB 80-I[-1282-1283, 92-V- 1609-1611, 5 specimens, 27.9-39.5 mm SL; Liao-He at Liaoning Province, China. G. tungi: THB 202108056037-6038, 2 specimens, Xin-Ji- ang, an effluent of Poyang Lake at Shangrao City, Ji- angxi Province, China. Xenophysogobio boulengeri. MNHN-IC-1934-0180, holotype, Szechwan (now Sichuan Province), China (X-radiograph and photograph examined); AMNH 20522, 2 specimens, 30.0—-31.2 mm SL; Yueyang City, Hunan Province, China (photograph examined); THB78IV0215-16, 0218, 3 specimens, Min-Jiang, an zse.pensoft.net Chen, X. et al.: Gobiobotia Iii and G. nicholsi effluent of the upper Chang-Jiang at Leshan, Sichuan Province, China. Ethics approval and consent to participate All procedures described in this paper were in accor- dance with Chinese laws and were licensed by the Min- istry of Ecology and Environment of the People’s Re- public of China. Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on rea- sonable request. Competing interests The authors declare that they have no competing interests. Funding This study was granted by special fund of Program for Biodiversity Investigation & Assessment Project for Biodiversity Conservation of Lake Dongting (Grant No. 2016HB2096001006) and National Science & Technol- ogy Fundamental Resources Investigation Program of Mount Dabie (Grant No. 2019FY 101800). Authors’ contributions Xiao Chen and Man Wang conceived the study and anal- ysed the data. Xiao Chen led the writing. Liang Cao and E Zhang revised the manuscript. All authors contributed to the writing of the paper. Acknowledgements Our sincere thanks should be given to Prof. Jian-Zhong Shen (HZAU, Huazhong Agricultural University) and Hong Li (Hunan Fisheries Science Institute) for assisting us in field- works and Wei-Han Shao, Zi-Tong Wang, Dong-Ming Guo, Xiong Gong and Yi Liu (THB) for fieldworks and laboratory analysis. Special thanks go to Chang-Ting An (IHB), who provided constructive suggestions for this manuscript. We thank Radford Arrindell (AMNH) and Peter Bartsch and Edda ABel (ZMB) for friendly help in providing specimen photographs and X-radiographs. We greatly appreciated Prof. Sven Kullander, Dr. Fan Li and Anonymous Reviewer for constructive comments to improve our manuscript. Zoosyst. Evol. 98 (1) 2022, 93-107 References Avise JC, Walker D (1999) Species realities and numbers in sexual ver- tebrates: Perspectives from an asexually transmitted genome. Pro- ceedings of the National Academy of Sciences of the United States of America 96(3): 992-995. https://doi.org/10.1073/pnas.96.3.992 Banarescu P, Nalbant TT (1966) Notes on the genus Gobiobotia (Pisces, Cyprinidae) with description of three new species. Annotationes Zoologicae et Botanicae 27: 1-16. Chen Y, Cao W (1977) Gobiobotinae. Fish of Chinese Cyprinidae (I). Shanghai Scientific & Technical Publishers, Shanghai, 550-570. Ding R (1994) The fishes of Sichuan, China. 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