Introduction

The genus Triplophysa Rendahl is a large group of loaches in the family Nemacheilidae of order Cypriniformes, which comprises over 180 valid species or subspecies distributed in the Qinghai-Tibet Plateau and adjacent regions (Zhu 1989, Eschmeyer et al. 2022). Species of Triplophysa are further subdivided into two groups based on their living habits and life-history traits: the epigean group and the hypogean group. Till now, 33 hypogean species of Triplophysa have been described, mainly found in the limestone caves or underground rivers of karst areas in southwestern China (Lan et al. 2013, Zhang et al. 2020, Chen et al. 2021, Deng et al. 2022). Meanwhile, the monophyly of both ecological groups of Triplophysa was also supported by recent phylogenetic analyses (Chen & Peng 2019, Chen et al. 2021).

Guizhou Province is located in southwestern China and has been recognised as a hotspot for cavefishes (Zhao & Zhang 2009). Nine hypogean species related to Triplophysa have been described in Guizhou Province, of which six are now valid, namely T. nasobarbatula Wang & Li, 2001 and T. zhenfengensis Wang & Li, 2001, T. longliensis Ren, Yang & Chen, 2012, T. guizhouensis Wu, He, Yang & Du, 2018, T. baotianensis Li, Liu, Li & Li, 2018, T. sanduensis Chen & Peng, 2019. Notably, all of the known Triplophysa species from Guizhou were captured from the River Pearl drainage. In addition, a recent ichthyological survey yielded two hypogean species of Triplophysa from the River Wujiang, a tributary of the upper River Yangtze in Guizhou Province, which could not be assigned to any of the other recorded species and are herein described as new species.

Material and Methods

Fish specimens were collected by dipnet. After anaesthesia, a small piece of muscle tissue was dissected from the right side of the dorsum and preserved in 95% ethanol for DNA extraction. The specimens were fixed in 10% formalin and later transferred to 5% formalin for long-term preservation. Morphological measurements were made point-to-point with a dial calliper and recorded to the nearest 0.1 mm from the left side of the specimens whenever possible. All measurements, counts and terminology follow Kottelat (1984). Inter-nostril width was regarded as the distance between both anterior nostrils. One specimen of each species was dissected to examine the intestine shape, the chambers of the air bladder, and the inner gill rakers. Vertebrae counts were conducted using images of micro-computed tomography (Siemens Somatom Definition X-ray machine) reconstructed in CTvox software. Comparative specimens of this study were deposited at the Institute of Hydrobiology, Chinese Academy of Sciences (IHB) in Wuhan, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ) in Kunming, Guizhou Normal University (GZNU) in Guiyang, and Southwest University (SWU) in Chongqing.

Genomic DNA was extracted from ethanol-preserved muscle tissues using a modified salt-extraction method (Tang et al. 2008). Mitochondrial cytochrome b (cytb) gene sequences were amplified by polymerase chain reaction (PCR) with the primers L14724 (5'-GACTTGAAAAACCACCGTTG-3') and H15915 (5'-CTCCGATCTCCGGATTACAAGAC-3') (Xiao et al. 2001). Amplifications were performed in a 30 µL reaction volume, containing 3 µL 10 × PCR buffer, 30-50 ng DNA template, 1 µL each primer (each 10 µM), 1.5 µL dNTPs (each 2.5 mM), and 2.5 U Taq DNA polymerase. Sterile water was added to reach the final volume. PCR procedures followed Tang et al. (2008). The amplified products were sent to a commercial sequencing company for purifying and sequenced in both directions with the same primers mentioned above.

Fig. 1.

Triplophysa wudangensis, holotype, IHB 2019080901 53.4 mm SL; from a subterranean tributary of the River Wujiang in the River Yangtze drainage in Wudang Distinct, Guiyang City, Guizhou Province, China. A) lateral, dorsal and ventral views; B) lateral and dorsal views of the head; C) lateral and ventral views of the micro-CT graph.

Phylogenetic analysis was performed based on four newly generated cytb sequences of the two new species and already published data from NCBI GenBank for additional 27 sequences, including 24 formally described Triplophysa species and two species of Homatula (outgroup) (Table 1). The sequences were aligned with ClustalX v2 (Larkin et al. 2007) and revised manually with SEAVIEW (Galtier et al. 1996). The Maximum Likelihood (ML) method was employed to reconstruct the phylogenetic relationship in IQ-TREE v1 (Nguyen et al. 2015), with optimal nucleotide substitution model (GTR + F + I + G4) based on Bayesian inference criterion; nodal support values were estimated from 1,000 nonparametric bootstrap replicates. Calculation of pairwise genetic distances based on the Kimura-2parameter (K2P) model (Kimura 1980) was conducted in MEGA v7 (Kumar et al. 2016).

Table 1.

GenBank accession numbers of included species in the molecular phylogenetic analysis.

Results

Table 2.

Morphometric characters of Triplophysa wudangensis, T. qingzhenensis and T. rosa. Ranges include values of holotype and paratypes. SD – standard deviation.

Fig. 2.

Live photo of A) Triplophysa wudangensis; B) Triplophysa qingzhenensis.

Fig. 3.

Habitat of Triplophysa wudangensis.

Fig. 4.

Type localities of hypogean species of Triplophysa in Guizhou Province and the River Yangtze drainage.

Table 3.

Major diagnostic characters for hypogean species of Triplophysa. % HL – percentage of head length; P – pectoral fin; D – dorsal fin; V – ventral fin (pelvic fin); A – anal fin; C – caudal fin; n/a – not available.

Fig. 5.

Triplophysa qingzhenensis, holotype, IHB 2019111501 103.9 mm SL; from a subterranean tributary of the River Wujiang in the River Yangtze drainage in Qingzhen County, Guiyang City, Guizhou Province, China. A) lateral, dorsal and ventral views; B) lateral and dorsal views of the head; C) lateral and ventral views of the micro-CT graph.

Fig. 6.

Habitat of Triplophysa qingzhenensis. A) entrance to the subterranean tributary; B) the pool where type specimens were collected.

Discussion

There is an increasing number of cave species of Triplophysa described in the karst areas in the River Yangtze drainage. The descriptions of these two new species bring the total number to seven: T. rosa Chen & Yang, 2005, T. wulongensis Chen, Sheraliev, Shu & Peng, 2021, T. qini Deng, Wang & Zhang, 2022, T. xiangxienesis Yang, Yuan & Liao, 1986, T. erythraea Liu & Huang, 2019. from Yuanjiang system of Lake Dongting. Triplophysa wudangensis and T. qingzhenensis are significantly distinguishable by eye diameter (5.1-6.5% HL in T. wudangensis vs. 2.1-4.4% HL in T. qingzhenensis), interorbital width (33.1-35.8% HL in T. wudangensis vs. 25.1-30.4% HL in T. qingzhenensis), and vertebrae (4 + 34 in T. wudangensis vs. 4 + 36 in T. qingzhenensis). Triplophysa rosa (Fig. 8) was the first described cavefish from the River Wujiang system (Chen & Yang 2005), which was the closest relative to T. wudangensis and T. qingzhenensis inferred from intraspecies genetic distances. The morphometric characters of T. rosa are summarised in Table 2. The two new species distinctly differ from T. rosa by the presence of reduced eyes (vs. eye absent), a shorter pectoral fin (18.6-19.7% SL in T. wudangensis and 17.1-19.3% SL in T. qingzhenensis vs. 25.5% SL in T. rosa), distal margin of dorsal fin truncate (vs. concave), less branched dorsal-fin rays (7 in T. wudangensis and 7-8 in T. qingzhenensis vs. 9 in T. rosa), less branched anal-fin rays (5 in T. wudangensis and T. qingzhenensis vs. 6 in T. rosa). The two species are distinct from T. qini, T. xiangxiensis and T. erythraea by the presence of reduced eyes (vs. eye absent) and pelvic-fin tip not reaching to the anus (vs. reaching to); further differ from T. qini by the presence (vs. absence) of a barbel-like tip on the anterior nostril, and distal margin of pelvic fin truncate (vs. concave); from T. xiangxiensis by the posterior chamber of the air bladder degenerated (vs. developed), pectoral-fin tip not reaching to pelvic-fin origin (vs. reaching to), distal margin of dorsal fin truncate (vs. concave), and branched anal-fin rays 5 (vs. 6); from T. erythraea by the posterior chamber of air bladder degenerated (vs. developed), presence (vs. absence) of a barbel-like tip on the anterior nostril, branched anal-fin rays 5 (vs. 6), and branched caudal-fin rays 14 (vs. 16). Triplophysa wulongensis is a unique hypogean species in the River Yangtze drainage with normal-sized eyes (eye diameter 11.1-19.1% HL). The two species are further distinct from T. wulongensis by shorter interorbital width (33.1-35.8% HL in T. wudangensis and 25.1-30.4% HL in T. qingzhenensis vs. 38.5-43.1% HL in T. wulongensis), distal margin of dorsal fin truncate (vs. concave), branched caudal-fin rays 14 (vs. 18) and vertebrae (4 + 34 in T. wudangensis and 4 + 36 in T. qingzhenensis vs. 4 + 38 in T. wulongensis).

Molecular phylogenetic evidence corroborated the validities of T. wudangensis and T. qingzhenensis by the highly supported monophyly and sufficient genetic divergence from their congeners. Furthermore, the genetic distances between both species and their closest congener, T. rosa, were slightly less than the 2% threshold value, implying the differentiation of these species was at an early stage. Meanwhile, it should be noted that T. rosa, T. qini, T. xiangxiensis and T. erythraea are more analogous in morphology (eyes absent, loss of pigmentation, for instance), while the topology of the phylogenetic tree demonstrated that these hypogean species clustered neither by the river-system pattern nor by the morphological analogousness. On the one hand, this phenomenon was possibly caused by convergent evolution under similar selection pressure to other cave species (Wilkens & Strecker 2003, Xiao et al. 2005). On the other hand, it can be inferred that these hypogean species of Triplophysa may originate from heterogenetic ancestors and spread across different river systems. To be specific, the diversified hypogean species originated from the River Pearl drainage may disperse to the Wujiang system through subterranean river capture and connection and then spread to the Wuling Mountain area in Chongqing City and Hunan Province, as Wujiang is the largest river in Guizhou Province with concentrative-distributed and well-developed karst landform (Che & Yu 1985). A similar conclusion has also been drawn in the study on the dispersal process of Sinocyclocheilus cyphotergous species complex in the River Yangtze drainage (Wen et al. 2022). Therefore, the evolutionary history and distribution pattern of hypogean Triplophysa species were rather complex. A detailed field survey in Wujiang and the adjacent drainages is warranted to clarify the diversity pattern of this taxon in the River Yangtze drainage.

Fig. 7.

Maximum likelihood tree derived from cytb gene sequences of 26 species of Triplophysa. Numbers below the branches represent bootstrap values (> 50 shown).

Due to sampling difficulties, the current distribution range and population sizes of T. wudangensis and T. qingzhenensis remain unclear, while most reported cavefishes are deemed endemic to a narrow region, even a single cave (Zhao & Zhang 2006). In the survey, we found anthropogenic activities posed threats to the two species in their type localities. For T. wudangensis, the subterranean river has been utilised as a water source for a mineral water factory; and for T. qingzhenensis, factories have discharged sewage into the groundwater. Domestic pollution, illegal fishing and alien invasion (Gambusia affinis, Procambarus clarkii and Rana catesbiana, for instance) also threaten these two species. Guiyang City and its surrounding area is the type locality for many cavefishes, as well as endemic fishes of the River Yangtze (Zeng et al. 2022), which plays essential roles in biodiversity conservation and biogeographical research. At the same time, it is the most urbanised area of Guizhou Province. Therefore, environmental conservation measures are urgently needed to protect these species and their habitats.

Fig. 8.

Lateral, dorsal and ventral views of Triplophysa rosa, holotype, KIZ 200211001, holotype, 56.4 mm SL (photo Rui Min).

Comparative Materials

Triplophysa baotianensis: GZNU 20190524001, 69.08 mm SL; a tributary of the River Pearl at Baotian Town, Panzhou City, Guizhou Province.

Triplophysa guizhouensis: IHB 201906180001, GZNU 20200806001-20200806003, 4 specimens, 64.7-92.9 mm SL; a tributary of the Mengjiang system in the River Pearl drainage at Jialie Village, Huishui County, Guizhou Province.

Triplophysa nasobarbatula: GZNU 201731004-201731006, 3 specimens, 64.7-92.9 mm SL; a tributary of the Liujiang system in the River Pearl drainage at Yaosuo Village, Libo County, Guizhou Province.

Triplophysa qini: IHB 201809049887, holotype, 76.8 mm SL; Erwang Cave of the Wujiang system in the River Yangtze drainage at Houping Village, Wulong County, Chongqing City.

Triplophysa rosa: KIZ 200211001, holotype, 56.4 mm SL; Dongba Cave, of the Wujiang system in the River Yangtze drainage at Tianxing Town, Wulong County, Chongqing City.

Triplophysa sanduensis: SWU 2017061301-2017061304, paratypes, 4 specimens, 45.2-71.5 mm SL; a cave of the Liujiang system in the River Pearl drainage at Dengguang Village, Zhonghe Town, Sandu County, Guizhou Province.

Triplophysa xiangxiensis: IHB 2015010001, IHB 2015010004, IHB 2015010006, IHB 2015010009, topotypes, 4 specimens, 48.3-85.7 mm SL; Feihu Cave of the River Yuanjiang in the River Yangtze drainage at Huoyan Village, Longshan County, Hunan Province.

Triplophysa zhenfengensis: GZNU 20190514002, 79.0 mm SL; a tributary of the River Beipanjiang in the River Pearl drainage at Gaoyan village, Xingren City, Guizhou Province.

Comparative data were acquired from literatures for the following species: T. aluensis from Li & Zhu (2000); T. anshuiensis from Wu et al. (2018); T. erythraea from Huang et al. (2019); T. fengshanensis from Lan et al. (2013); T. flavicorpus from Yang et al. (2004); T. gejiuensis from Chu & Chen (1979); T. huapingensis from Zheng et al. (2012); T. langpingensis from Lan et al. (2013); T. longipectoralis from Zheng et al. (2009); T. longliensis from Ren et al. (2012); T. luochengensis from Li et al. (2017a); T. macrocephala from Yang et al. (2012); T. nandanensis from Lan et al. (1995); T. nanpanjiangensis from Zhu & Cao (1988); T. posterodorsalus from Ran et al. (2006); T. qiubeiensis from Li et al. (2008); T. shilinensis from Chen et al. (1992); T. tianeensis from Chen et al. (2004); T. tianlinensis from Li et al. (2017b); T. tianxingensis from Yang et al. (2016); T. wulongensis from Chen et al. (2021); T. xiangshuingensis from Li (2004); T. xichouensis from Liu et al. (2017); T. xuanweiensis from Lu et al. (2022); T. yunnanensis from Chu & Chen (1990).

Table 4.

Kimura-2-Parameter genetic distance (in %) among 14 cave species of Triplophysa based on cytb gene sequences.