All examined specimens were collected by hand net, fish trap, or bought from the local markets. Detailed information on the specimens is listed in the section Comparative material. Specimens used for morphological study were fixed in 10% formalin solution for three days, followed by 70% ethanol alcohol for long-term preservation. Specimens used for molecular phylogenetic study were fixed in 95% ethanol. The holotype of the new species was deposited at the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (ASIZB; abbreviation follows Leviton et al. 1985), paratypes were deposited at ASIZB, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China (IHB), and Laboratory of Ichthyology, Shanghai Ocean University, Shanghai, China (SHOU). The holotype of M. tungtingensis is stored at American Museum of Natural History, New York, the United States (AMNH). The syntypes of M. elongatus are stored at IHB. Other comparative materials are also deposited at ASIZB, IHB, and SHOU.

Measurements were taken point-to-point with a digital caliper to 0.01 mm on the left side of the specimens, and counts were also made on the left side of specimens. In order to make a more accurate measurement for lips structure, photographs of the lip papillae system were taken and images were analyzed with ImageJ v. 1.52 software. Once the scale was settled, the distance between two points was measured with a straight line (Lunghi et al. 2019). Counts and measurements followed Sun et al. (2021). In order to get a general perception on external morphologic differences, 30 different measurable traits, log₁₀-standardized to eliminate the allometries, were input into Past v. 4.03 (Hammer et al. 2001) for principal component analysis (PCA).

Molecular phylogenetic studies were based on the mitochondrial Cytochrome-b (Cyt-b) sequences. DNA was extracted from the pelvic fin on the right side of the fish. Cyt-b was amplified using the primers cytbF1 (5’-TGACTTGAAGAACCACCGTTGTA-3’) and cytbR1 (5’-CGATCTTCGGATTACAAGACCGATG-3’) following Huang et al. (2016). Sequencing reactions were performed according to the operating instructions of BigDye Terminator v. 3.1 (BDT), with 1 μL of primer (3.2 pmol/μL), 1 μL of template DNA, 2 μL of BigDye® Terminator v. 3.1, and 6 μL of double distilled water (dd H2O) for a total reaction volume of 10 μL. The thermo-cycling conditions were initial denaturation for 2 min at 96 °C, denaturation for 10 s at 96 °C, annealing for 10 s at 50 °C, and extension for 1 min at 60 °C. After 30 cycles, a final extension was performed at 60 °C for 3 min and the polymerase chain reaction (PCR) products were preserved at 4 °C. Sequencing was conducted by Beijing TsingKe Biotech Co., Ltd. (China).

The sequencing results were assembled using SeqMan II, and other sequences were acquired from the NCBI database. The voucher ID of each individual and GenBank accession No. are given in Table 1. Forty-three Cyt-b sequences of Microphysogobio species were included in the molecular phylogenetic analyses. Pseudogobio guilinensis was used as outgroup. Nucleotide sequence alignment was conducted using MEGA v. 6.0 (Tamura et al. 2013) with ClustalW. ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fit model using Bayesian information criterion (BIC). The Bayesian inference (BI) phylogenies were inferred using MrBayes v. 3.2.6 (Ronquist et al. 2012) under the HKY+F+G4 model (two parallel runs, 1 000 000 generations), with the initial 25% of sampled data discarded as burn-in. Maximum likelihood (ML) phylogenetic analysis was conducted using MEGA 6.0 under the HKY+G+I model (10 000 bootstrap replications). Trees were visualized by using TVBOT (https://www.chiplot.online/tvbot.html; Xie et al. 2023). Each sequence was labeled with its own taxonomic nomenclature and the evolutionary divergence of sequence pairs between and within groups (i.e., species) was estimated using the Kimura 2-parameter model (Kimura 1980). In addition, two independent methods, i.e., Poisson tree process (PTP) and assemble species by automatic partitioning (ASAP), which rely on different operational criteria, were applied to infer molecular species delineation for Microphysogobio (Kapli et al. 2017; Puillandre et al. 2020). The rooted phylogenetic trees (both BI and ML) were uploaded to the PTP online server (http://species.h-its.org/ptp/). Aligned sequences were uploaded to the ASAP online server using the Jukes-Cantor (JC69) model (https://bioinfo.mnhn.fr/abi/public/asap/). The delineation results were visualized on the trees, respectively.

Microphysogobio elongatus was originally described as a slender (body depth less than 16.7% of SL) species with six branched anal-fin rays and distribution in the Xijiang River, the tributary of the Pearl River basin (Luo et al. 1977). In its original description, it can be distinguished from “M. tungtingensis” (based on three non-types) only by their branched anal-fin rays (six rays in M. elongatus vs five rays in “M. tungtingensis”) and their distribution. According to the subsequent studies which followed Luo et al. (1977), the key character between M. tungtingensis and M. elongatus is still basically on branched anal-fin ray count. However, after carefully checking the original descriptions of M. tungtingensis along with the other Microphysogobio species described by Nichols himself (Nichols 1926a, 1926b, 1930) and examining the X-ray photos of those types, we confirmed that the branched anal-fin rays of M. tungtingensis should be six. Thus, it is not easy to distinguish M. tungtingensis and M. elongatus in this situation but only by their distribution (former in the Lake Dongting system in the middle Yangtze River basin vs latter in the Xijiang River system of the Pearl River basin), causing the validity of the latter suspicious.

In order to understand the potential morphological differences between these two species, 76 specimens, including the types of both species, from a wide geographical range, were examined (Figs 1, 7). The result shows that there is no significant difference in both counts and measurable traits (Table 2). The PCA on those morphometric measurements were performed to make comparisons in detail. The first three component contributed 82.3% of the variance. Principle component 1 (PC 1) represents most on the body size of the specimens, while PC 2 and PC 3 reflected their morphology. The PCA loadings are presented in Table 4, showing that the lip papillae system pattern contributes most in PC 2 and PC 3. However, both PC1 against PC2 and PC2 against PC3 are not able to separate these two species (Fig. 8).

Figure 7. 

Microphysogobio elongatus (= M. tungtingensis), syntype, IHB 58-7-876, 77.2 mm SL A lateral view B dorsal view, and C ventral view.

Figure 8. 

Scatterplot of the PC1 against PC2 (A) and PC2 against PC3 (B) extracted from 30 morphometric data of Microphysogobio tungtingensis and M. elongatus. Letter “T” marked inside the dots indicate the type specimens.

Molecular phylogenetic analyses were also conducted. The intraspecific distance of M. tungtingensis and M. elongatus was 0.16% and 0.04% respectively, while the interspecific distance between them is 0.13%. This value is approximate to the intraspecific distance of M. tungtingensis, making it hard to tell they are different. Besides, the interspecific distance is far less than any other showed in the genus Microphysogobio. The phylogenetic trees (Fig. 9) also demonstrate undetectable length of the branches.

Figure 9. 

Molecular phylogenetic trees of Microphysogobio punctatus sp. nov., M. tungtingensis, M. elongatus and other phylogenetically close related congeners based on Cyt b sequence (1140 bp) A Bayesian Inference method B maximum likelihood method.

Although distributed in different river basins, such distribution might be interpreted by two previously suggested hypotheses. One is that the Lingqu Canal, an ancient artificial canal connected in 214 B.C. during Qin Dynasty which connecting the Xiangjiang River with the Lijiang River, might cause the trans-basin gene flow between the Yangtze River basin and the Pearl River basin, which was discovered for M. zhangi (Huang et al. 2017). This interpretation was supported by the shared haplotype (Table 1) from both river basins. The second hypothesis is the episodic river capture events between the interwoven watersheds of the Nanling Mountains’ complex landform, e.g., the Lijiang River capture (Ru 2011), causing the cross-basin distribution, which was found in Sarcocheilichthys parvus (Li et al. 2023). Given the low intraspecific and interspecific distances (Table 5) within the phylogenetic linage M. tungtingensis located (M. tungtingensis + M. elongatus + M. fukiensis + M. kiatingensis) and their broad geographical distribution (from the upper Yangtze River basin to the southeast coastal river systems), we drew the conclusion that the species within this linage performed a relatively conserved mutation in molecular. Hence, according to the trees shown in Fig. 9, it is also likely that the cross-basin distribution was caused by the very recent river capture event which was not long enough ago to accumulate a certain mutation to be detected by the current mitochondrial gene data, especially in this conserved linage.

Based on the morphological comparisons, molecular phylogenetic analyses, and its distribution, we suggest M. elongatus to be a junior synonym of M. tungtingensis. On the basis of the revision of M. tungtingensis, we are now able to distinguish the new species, with an overlapping distribution, discussed below.

Among the 30 known Microphysogobio species, M. punctatus sp. nov. can be distinguished from M. brevirostris (Günther, 1868), M. chinssuensis (Nichols, 1926), M. yaluensis (Mori, 1928), M. hsinglungshanensis Mori, 1934, M. koreensis Mori, 1935, M. longidorsalis Mori, 1935, M. amurensis (Taranetz, 1937), M. alticorpus Bănărescu & Nalbant, 1968, M. anudarini Holcík & Pivnicka, 1969, M. liaohensis (Qin, 1987), M. rapidus Chae & Yang, 1999, M. jeoni Kim & Yang, 1999, M. wulonghensis Xing, Zhao, Tang & Zhang, 2011, M. nudiventris Jiang, Gao & Zhang, 2012, M. xianyouensis Huang, Chen & Shao, 2016, and M. oujiangensis Sun & Zhao, 2022 by having a narrower upper jaw cutting edge (width 22.0%–46.8% of mouth width vs larger than half of mouth width).

For the remaining 14 congeners, the new species can be distinguished from M. linghensis Xie, 1986 and M. microstomus Yue, 1998 by having well developed lip papillae system (vs undeveloped or less developed papillae on lips, Fig. 3E). It can be distinguished from M. kiatingensis (Wu, 1930) by having a larger squamous zone on mid-ventral region (scaleless only before pectoral-fin base end vs scaleless before two-thirds of the distance from the pectoral-fin insertion to the pelvic-fin insertion). It can be distinguished from M. bicolor (Nichols, 1930), M. tafangensis (Wang, 1935), M. zhangi Huang, Zhao, Chen & Shao, 2017 by having more branched anal-fin rays (6 vs 5). For M. zhangi, the species sympatric with the new species, it can be further distinguished from the new species by having different anterior papillae pattern on upper lip (two large papillae vs two to six similar sized papillae, Fig. 3D, F) It can also be distinguished from M. fukiensis (Nichols, 1926), M. tungtingensis (Nichols, 1926) and M. vietnamica Mai, 1978 by having larger posterior chamber of the air-bladder (length 58.6%–82.8% of eye diameter vs length 21.9%–43.1%, 30.4%–34.9% and 35.5%–64.2% of eye diameter respectively). Considering M. vietnamica has overlap on air-bladder size with the new species, it can be further distinguished from latter by having different anterior papillae pattern on upper lip (two large papillae separated from each other vs similar sized papillae tightly contacted with each other, Fig. 3D, G). The new species can be distinguished from M. pseudoelongatus Zhang & Zhao, 2001 by having more circumpeduncular scales (12 vs 10). For the M. nikolskii (Dao & Mai, 1959), the species inquirenda (Kottelat 2013), the new species can be distinguished from it by having fewer lateral-line scales (37–40 vs 43; Huang et al. 2018).

For the rest of the three congeners, they are morphologically similar with the new species by having a larger posterior chamber of the air-bladder (larger than half eye diameter and sometimes equal to eye diameter) and tightly contacted central portion of anterior papillae. The new species can be distinguished from these three species by having more black pigmentation on fin rays. In addition, the new species can be distinguished from Microphysogobio yunnanensis (Yao & Yang, 1977) by having fewer scales above lateral line (3.5–4 vs 4.5), from M. luhensis Huang, Chen, Zhao & Shao, 2018 by having a larger eye diameter (30.7%–38.5% vs 25.2%–27.9% of head length), and from M. kachekensis (Oshima, 1926) by having a more slender caudal peduncle (depth 34.6%–48.5% vs 48.9%–55.7% of length).

As for Microphysogobio bicolor, although it has already been distinguished from the new species by having fewer branched anal-fin rays (5 vs 6), it is also morphologically close to the new species by having similar size of the air-bladder (63.1%–82.0% vs 50.3%–82.8% of eye diameter in average) and lip papillae pattern (Fig. 3H). However, the new species has a slender caudal peduncle (depth 34.6%–48.5% vs 43.3%–58.4% of length; Fig. 10) and shorter preanal distance (71.5%–78.1% vs 75.5%–90.0% of SL) as compared to M. bicolor.

Figure 10. 

Morphometric measurement divergence in caudal peduncle depth between Microphysogobio punctatus sp. nov. and M. bicolor.

A total of 37 haplotypes from 43 Microphysogobio individuals for Cyt b gene were included in the molecular phylogeny study. Based on molecular phylogenetic analyses, the new species is sister to M. bicolor, and together sister to M. luhensis–M. kachekensis–M. yunnanensis clade. The interspecific distance between M. punctatus and M. bicolor is 6.65% for Cyt b based on K2P model. The intraspecific distance within M. punctatus is 3.24% based on K2P model, which is lower than the interspecific distance. In fact, the molecular species delineation method (ASAP and PTP) inferred that the three geographic populations (Lingchuan, Yongfu, and Guanyang counties) to be three different molecular species which forming a monophyletic lineage. However, there is no significant difference in morphology among three populations, plus the closely connected river system within their distribution, we treated them as one species.

Microphysogobio bicolor : • AMNH 9678, holotype, 58.7 mm SL; Hekou Township, Yanshan County, Shangrao City, Jiangxi Province, from the Xinjiang River; collected by Clifford H. Pope; 22 June 1926 • ASIZB 220619–27, 9 specimens, 39.1–50.3 mm SL; Yongping Township, Yanshan County, Shangrao City, Jiangxi Province, from the Yanshanhe River (28.21615237°N, 117.79050896°E, 75 m a.s.l.); collected by Zhixian Sun and Rui Zhang; 11–12 April 2021 • ASIZB 213554, 1 specimen, 52.4 mm SL; Yongping Township, Yanshan County, Shangrao City, Jiangxi Province, from the Yanshanhe River (28.21615237°N, 117.79050896°E, 75 m a.s.l.); collected by Zhixian Sun, Rui Zhang, Chen Tian, and Xin Wang; 20 July 2021 • ASIZB 220628–35, 8 specimens, 42.1–62.2 mm SL; Taibai Township, Wuyuan County, Shangrao City, Jiangxi Province, from the Le’anhe River (29.07164455°N, 117.67277022°E, 47 m a.s.l.); collected by Junhao Li and Zhixian Sun; 16 December 2020 • ASIZB 220636–40, 220647, 220650, 220652, 220654, 9 specimens, 63.1–84.5 mm SL; bought from the Dongmen Market from the local fishermen, Wuyuan County, Shangrao City, Jiangxi Province, from the Le’anhe River; collected by Zhixian Sun and Rui Zhang; 11 April 2020 • ASIZB 229848–9, 2 specimens, 59.0–76.0 mm SL; bought from the Wangjiaba Market from the local fishermen, Guangxin District, Shangrao City, Jiangxi Province, from the Xinjiang River; collected by Zhixian Sun, Rui Zhang, Chen Tian, and Xin Wang; 21 July 2021.

Microphysogobio fukiensis : • ASIZB 220660–68, 9 specimens, 50.5–64.6 mm SL; bought from the local market, Guangze County, Nanping City, Fujian Province, from the Futunxi River; collected by Zhixian Sun, Rui Zhang, Qiuju Chen and Rui Xi; 17 April 2021.

Microphysogobio kechekensis : • ASIZB 240478–88, 11 specimens, 64.8–83.6 mm SL; bought from the local market, Baisha Li Autonomous County, Hainan Province, probably caught by fishermen from the Wanquanhe River; collected by Shanyu Wu; 3 April 2020 • ASIZB 69768, 1 specimen, 100.0 mm SL; Gongzheng Township, Shangsi County, Fangchenggang City, Guangxi Zhuang Autonomous Region, from a stream of Maolingjiang River; collected by Yahui Zhao; 29 April 1999.

Microphysogobio luhensis : • ASIZB 204717, paratype, 55.5 mm SL; Dongkeng Township, Luhe County, Shanwei City, Guangdong Province, from the Rongjiang River (23.30165309°N, 115.71898652°E, 126 m a.s.l.); collected by Shih-Pin Huang; 2 April 2009.

Microphysogobio microstomus : • SHOU 20231209017, 47.3 mm SL; Shanhu Township, Shengzhou City, Shaoxing City, Zhejiang Province, from the Shanxi River (29.63724853°N, 120.84212848°E, 12 m a.s.l.); collected by Zhixian Sun; 9 December 2023.

Microphysogobio vietnamica : • ASIZB 240561–71, 11 specimens, 43.0–53.5 mm SL; Shixing County, Shaoguan City, Guangdong Province, from the Mojianghe River; collected by Zhixian Sun; 4 January 2021.

Microphysogobio yunnanensis : • IHB 645480, 645486–7, 645492, 645494–5, 645499, syntypes, 7 specimens, 73.8–82.2 mm SL; Yuanjiang Hani Yi and Dai Autonomous County, Yuxi City, Yunnan Province, from the Yuanjiang River (the Red River); May 1964 • IHB 645496, 6440132, 6440136, 6440296, 6440638, 6440648–9, 6440654, 6450063, syntypes, 9 specimens, 68.0–95.9 mm SL; Hekou Yao Autonomous County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, from the Yuanjiang River (the Red River); April and May 1964.

Microphysogobio zhangi : • ASIZB 204677, holotype, 75.1 mm SL; Quanzhou County, Guilin City, Guangxi Zhuang Autonomous Region, from the Xiangjiang River (25.93332646N, 111.08037923E, 151 m a.s.l.); collected by Shih-Pin Huang and Jinqing Huang; 6 November 2015 • ASIZB 220669–76, 8 specimens, 48.2–63.6 mm SL; Pingle County, Guilin City, Guangxi Zhuang Autonomous Region, from the Lijiang River; collected by Zhixian Sun; 23 January 2021.

The authors have declared that no competing interests exist.

No ethical statement was reported.

This work was supported by the National Natural Science Foundation of China (NSFC-32270464) and Sino BON - Inland Water Fish Diversity Observation Network.

Sun ZX contributed to the field collection, photographing, illustrating, specimens examining, data analyses and drafting of the manuscript. Tang WQ and Zhao YH was responsible for review and editing.

Zhi-Xian Sun https://orcid.org/0000-0003-0565-2801

Wen-Qiao Tang https://orcid.org/0000-0001-5992-5022

Ya-Hui Zha https://orcid.org/0000-0002-4615-596X

All of the data that support the findings of this study are available in the main text or Supplementary Information.