Triplophysa wulongensis, a new species of cave-dwelling loach (Teleostei, Nemacheilidae) from Chongqing, Southwest China

Shijing Chen; Bakhtiyor Sheraliev; Lu Shu; Zuogang Peng

doi:10.3897/zookeys.1026.61570

Abstract

We describe a new species of cave-dwelling loach, Triplophysa wulongensis sp. nov., based on specimens collected in a subterranean pool in a cave in Wulong County, Chongqing, Southwest China. The pool is connected to the Wujiang River drainage. Triplophysa wulongensis differs from its congeners by the following combination of characters: eyes present, caudal fin with 18 branched rays; posterior chamber of the air bladder degenerate; stomach U-shaped; intestine without bends or loops immediately posterior to stomach; body smooth and scaleless, and lateral line complete. The mitochondrial cytochrome b sequence differs from those of other published sequences of species of Triplophysa by 14.9–24.9% in K2P distance. Phylogenetic analysis based on cytochrome b gene sequences recovered T. wulongensis as sister taxon to all other cave-dwelling species of Triplophysa.

Keywords

Cavefish, cytb sequence, freshwater fish, ichthyology, phylogeny

Introduction

The genus Triplophysa Rendahl, 1933, currently comprises approximately 160 valid species, most of which are known from Qinghai-Tibet Plateau and to a lesser extent from Central Asia (Zhu 1989; Prokofiev 2010; Kottelat 2012; Fricke et al. 2020). Triplophysa is distinguished from other genera of Nemacheilidae by a marked sexual dimorphism, in which males have tubercle-bearing, elevated skin on the side of the head, and a thickened tuberculated pad on the dorsal surface of the thickened and widened rays of the pectoral fin. Species of Barbatula Linck, 1790 share the same sexual dimorphism, but Triplophysa can be distinguished from Barbatula by the closely situated nostrils (Bănărescu and Nalbant 1968; Prokofiev 2010; Yang et al. 2012; Liu et al. 2017).

To date, 33 cave-dwelling species of Triplophysa have been described from the karst areas of southern China where karst caves and subterranean streams are dominant geological features (Lan et al. 2013; Liu et al. 2017; Wu et al. 2018a). According to Lan et al. (2013), these species can be placed into three groups based on the state of the eyes, namely, eyes normal, reduced, or absent (Table 1).

Table 1.

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Characters variable across cave-dwelling species of Triplophysa from China.

No	Species	Eyes	Scales	Lateral line	Posterior chamber of air bladder	Dorsal	Anal	Pectoral	Pelvic	Caudal	Tip of pelvic fin reaching anus	Anterior nostril barbel-like
No	Species	Eyes	Scales	Lateral line	Posterior chamber of air bladder	fin rays	fin rays	fin rays	fin rays	fin rays	Tip of pelvic fin reaching anus	Anterior nostril barbel-like
1.	T. aluensis	Reduced	Absent	Complete	Degenerated	iii, 7	iii, 5	i, 9	i, 6	13	No	Yes
2.	T. anshuiensis	Absent	Absent	Complete	Developed	iv, 7–8	ii, 6	i, 10	i, 6	14	Yes	Yes
3.	T. baotianensis	Normal	Absent	Complete	Degenerate	iii, 6–7	ii, 4–5	i, 9	i, 5	11–13	No	Yes
4.	T. erythraea	Absent	Absent	Complete	Developed	ii, 8	i, 6	ii, 10	ii, 5	17	Yes	No
5.	T. fengshanensis	Absent	Absent	Complete	–	ii, 8	ii, 6	i, 8–10	i, 6–7	16	No	Yes
6.	T. flavicorpus	Normal	Present	Complete	Degenerated	iii, 10	iii, 6–7	i, 11	i, 6–7	16	Yes	No
7.	T. gejiuensis	Absent	Absent	Complete	Developed	iii, 7–8	iii, 4–6	i, 10	i, 5	14–15	Yes	Yes
8.	T. guizhouensis	Normal	Present	Complete	Developed	iii, 8	iii, 6	i, 8–9	i, 6	14	No	Yes
9.	T. huanjiangensis	Absent	Absent	Absent	Developed	iii, 8–9	iii, 6–7	i, 10–14	i, 6–7	13–14	No	Yes
10.	T. huapingensis	Normal	Present	Complete	Degenerated	iii, 8–9	iii, 5	i, 9–10	i, 5–6	16	No	No
11.	T. langpingensis	Reduced	Absent	Incomplete	–	iii, 7–8	iii, 5–6	i, 10–11	i, 6	14	Yes	Yes
12.	T. lingyunensis	Reduced	Present	Incomplete	Degenerated	iii, 7–8	iii, 5	i, 8–9	i, 5–6	16	No	Yes
13.	T. longipectoralis	Normal	Present	Complete	Degenerated	iii, 8	iii, 5–6	i, 9–10	i, 6	14–15	Yes	Yes
14.	T. longliensis	Normal	Absent	Complete	Developed	iii, 8	iii, 5	i, 10	i, 6	15–16	Yes	Yes
15.	T. luochengensis	Reduced	Present	Complete	Degenerated	iii, 8	ii, 6	i, 10	i, 6	16–17	No	Yes
16.	T. macrocephala	Reduced	Absent	Complete	Degenerated	iii, 7–9	iii, 5–6	i, 9–11	i, 6	15–17	Yes	Yes
17.	T. maolanensis	Absent	Absent	Complete	–	iii, 8	ii, 5	i, 11	i, 6	14	Yes	No
18.	T. nandanensis	Normal	Present	Complete	Degenerated	iv, 8	iv, 5	i, 9–10	i, 6	14–16	No	Yes
19.	T. nanpanjiangensis	Normal	Absent	Complete	Degenerated	iii, 7–8	ii, 5	i, 9–10	i, 6	16	No	Yes
20.	T. nasobarbatula	Normal	Present	Complete	Degenerated	iii, 8	iii, 5	i, 9	i, 6	15	Yes	Yes
21.	T. posterodorsalus	Absent	Absent	Complete	–	iii, 6	ii, 4	i, 13	i, 5	15	No	Yes
22.	T. qiubeiensis	Absent	Absent	Complete	Degenerated	iii, 7	iii, 5	i, 7–9	i, 5	14–15	Yes	No
23.	T. rosa	Absent	Absent	Complete	–	iii, 9	iii, 6	i, 12	i, 7	14	Yes	Yes
24.	T. sanduensis	Normal	Present	Complete	Degenerated	ii, 8–9	i, 5	i, 8–9	i, 5	17–18	No	Yes
25.	T. shilinensis	Absent	Absent	Complete	Degenerated	iii, 7	iii, 5	i, 8–10	i, 6	14	No	Yes
26.	T. tianeensis	Reduced	Absent	Complete	Degenerated	iii, 6–7	iii, 5	i, 8–9	i, 5–6	15–16	No	Yes
27.	T. tianlinensis	Reduced	Absent	Complete	Degenerated	iii, 7	iii, 5–6	i, 10	i, 6	15–16	Yes	Yes
28.	T. tianxingensis	Normal	Absent	Complete	Developed	iii, 8	ii, 5	i, 9	i, 5	16	No	No
29.	T. wulongensis sp. nov.	Normal	Absent	Complete	Degenerated	ii, 8–9	i, 5–6	i, 8–9	i, 5–7	18	No	Yes
30.	T. xiangshuingensis	Normal	Absent	Complete	Degenerated	iii, 6	iii, 5	i, 9	i, 6	14	No	Yes
31.	T. xiangxiensis	Absent	Absent	Complete	Developed	iii, 8	iii, 6	i, 11	i, 6	16	Yes	Yes
32.	T. xichouensis	Reduced	Absent	Complete	Developed	iii, 8	ii, 6	i, 9–10	i, 5–6	16	Yes	Yes
33.	T. yunnanensis	Normal	Present	Complete	Degenerated	iii, 7	iii, 5	i, 9–10	i, 7	15–16	No	Yes
34.	T. zhenfengensis	Normal	Present	Complete	Degenerated	iii, 7	iii, 5	i, 9	i, 5–7	14–15	No	Yes

We collected nine loach specimens from a subterranean pool in a cave located in Wulong County, Chongqing, Southwest China. Morphological and molecular analyses justified the recognition of this sample as representing a new species of Triplophysa, described below.

Materials and methods

After anesthesia, the specimens were fixed in 10% formalin and stored in 70% ethanol. Measurements were made with digital calipers and rounded off to the nearest 0.1 mm. All measurements were made point to point, and whenever possible, measurements and counts were recorded on the left side of the body following the methods described by Kottelat and Freyhof (2007). The standard length was measured from the tip of the snout to the end of the hypural complex; the length of the caudal peduncle was measured from behind the base of the last ray of the anal fin to the end of the hypural complex at mid-height of the base of the caudal fin. The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins were counted as a single ray. Fin rays were counted using a stereo microscope. Vertebrae from five specimens were observed on X-radiographs. The specimens examined were deposited in the Southwest University School of Life Sciences (SWU) in Beibei, Chongqing, P. R. China. Abbreviations are defined as follows: SL, standard length; HL, head length; CLJH, Collection of Lan Jiahu (private collection); GIF, Guangxi Institute of Fisheries, Guangxi, China.

Data on Triplophysa aluensis Li & Zhu, 2000, T. gejiuensis (Chu & Chen, 1979), T. nanpanjiangensis (Zhu & Cao, 1988), T. qiubeiensis Li & Yang, 2008, T. shilinensis Chen & Yang, 1992, T. tianxingensis Yang, Li & Chen, 2016, T. xiangshuingensis Li, 2004 and T. yunnanensis Yang, 1990 are from Yang et al. (2016); T. baotianensis Li, Li, Liu & Li, 2018 and T. longliensis Ren, Yang & Chen, 2012 from Li et al. (2018); T. maolanensis (Li, Ran & Chen, 2006) and T. posterodorsalus (Li, Ran & Chen, 2006) from Li et al. (2006); T. anshuiensis Wu, Wei, Lan & Du, 2018, T. flavicorpus Yang, Chen & Lan, 2004, T. guizhouensis Wu, He, Yang & Du, 2018, T. luochengensis Li, Lan, Chen & Du, 2017 and T. tianlinensis Li, Li, Lan & Du, 2016 from Wu et al. (2018b); T. erythraea Liu & Huang, 2019 and T. xichouensis Liu, Pan, Yang & Chen, 2017 from Huang at al. (2019); and T. xiangxiensis (Yang, Yuan & Liao, 1986) from Yang et al. (1986). Other species used for comparative purposes were examined at CLJH, GIF, and SWU, China (Suppl. material 1: Table S1).

DNA extraction and PCR

Genomic DNA was extracted from ethanol-preserved fin tissue using a DNeasy Blood and Tissue Kit (QIAGEN, Shanghai, China). The primers used for PCR amplification of the mitochondrial cytochrome b (cytb) gene are described by Xiao et al. (2001). PCR amplifications were performed in a total volume of 25 μL consisting of 14.8 μL of dd H₂O, 2.0 μL of DNA template (50 ng/μL), 1.0 μL of each primer (10 μM), 2.5 μL of 10× PCR buffer, 1.5 μL of 25 mM MgCl₂, 2.0 μL of 2.5 mM dNTPs, and 0.2 μL of rTaq DNA polymerase (TaKaRa; Dalian, China). The PCR conditions used were as follows: an initial denaturation step at 94 °C for 4 min followed by 34 cycles of 30 s at 94 °C, 50 s at 50–56 °C and 80 s at 72 °C; with a final extension of 8 min at 72 °C.

Molecular data analyses

We sequenced partial cytb gene of T. longliensis, T. nandanensis Lan, Yang & Chen, 1995, T. sanduensis Chen & Peng, 2019, T. tianeensis Chen, Cui & Yang, 2004, and T. wulongensis and retrieved the cytb gene sequences for other species of Triplophysa from GenBank (Table 2). Barbatula nuda (Bleeker, 1864) and B. toni (Dybowski, 1869) were selected as outgroup. Alignment of the cytb sequences was performed using the Clustal W algorithm in MEGA7 (Kumar et al. 2016), with manual checks for inconsistencies. MEGA7 was also used to calculate Kimura’s 2-parameter genetic distances (K2P). For phylogenetic reconstructions, the datasets were analyzed based on Bayesian inference (BI) methodology using MrBayes 3.2 (Ronquist et al. 2012) and the maximum likelihood (ML) method of MEGA7 (Kumar et al. 2016). MrBayes used the Generalized Time Reversible model (nst = 6) and gamma-distributed rate variation and the proportion of invariable positions (GTR+G+I) for the cytb datasets. For BI, we ran four simultaneous Monte Carlo Markov chains for 2,000,000 generations, with sampling every 1,000 generations, and the first 25% of samples were discarded as burn-in. Tracer v. 1.7 (Rambaut et al. 2018) was used to assess convergence of the posterior, which was determined when effective sample size (ESS) values reached 200. For ML analyses, we conducted heuristic searches (1,000 runs) using a Kimura’s 2-parameter (K2P) model. The phylogenetic trees were visualized and edited using FigTree v. 1.4.2 (Rambaut 2014).

Table 2.

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The species used in this study with their GenBank accession number for the mitochondrial cytb gene sequences.

Species	GenBank accession number	Species	GenBank accession number
Barbatula nuda	KF574248	Triplophysa minxianensis	KT213596
Barbatula toni	AB242162	Triplophysa nandanensis	MW582824
Triplophysa anterodorsalis	KJ739868	Triplophysa rosa	JF268621
Triplophysa bleekeri	JQ686729	Triplophysa sanduensis	MW582822
Triplophysa brevicauda	KT213588	Triplophysa siluroides	KT213603
Triplophysa chondrostoma	KT213589	Triplophysa tianeensis	MW582826
Triplophysa erythraea	MG967615	Triplophysa tibetana	KT224364
Triplophysa huapingensis	MG697589	Triplophysa wulongensis	MW582823
Triplophysa lewangensis	KU987438	Triplophysa xiangxiensis	KT751089
Triplophysa longliensis	MW582825	Triplophysa xichangensis	KT224366
Triplophysa markehenensis	KT213594	Triplophysa zhenfengensis	MK610360
Triplophysa microps	KT213595

Results

Triplophysa wulongensis sp. nov.

http://zoobank.org/C5034BEA-EC81-4BC1-ADA8-E45CB1699B46

Figures 1, 2; Table 3

Type material

Holotype. SWU2019051309, male, 64.0 mm SL. P.R. China: Chongqing City; Wulong County: subterranean pool in Furong Cave (29°24'1.09"N, 107°54'11.60"E); collected by Ni Liu, May 2019.

Paratypes. SWU2019051301–2019051308, 8 ex., 49.0–67.2 mm SL; collected with the holotype.

Figure 1.

Triplophysa wulongensis sp. nov., holotype, SWU 2019051309, 64.0 mm SL A lateral view B dorsal view C ventral view.

Diagnosis

Triplophysa wulongensis can be distinguished from its congeners by the following combination of characters: eyes present (vs absent in T. anshuiensis, T. erythraea, T. huanjiangensis Yang, Wu & Lan, 2011, T. rosa Chen & Yang, 2005, T. xiangxiensis and T. posterodorsalus); anterior nostril barbel-like (vs anterior nostril not elongate to barbel-like in T. erythraea, T. flavicorpus, T. huapingensis Zheng, Yang & Chen, 2012 and T. tianxingensis): caudal fin with 18 branched rays (vs 14–16 in T. guizhouensis, T. lingyunensis (Liao, Wang & Luo, 1997), T. nandanensis, T. shilinensis and T. zhenfengensis Wang & Li, 2001), vertebrae 4+38–39 (vs 36–37 in T. nasobarbatula Wang & Li, 2001 and T. sanduensis; 42–43 in T. siluroides); predorsal length 50.4–54.2% of standard length (vs 46.1–48.0% in T. sanduensis); posterior chamber of gas bladder degenerate (vs developed in T. anshuiensis, T. tianxingensis and T. xichouensis); body smooth and scaleless (vs body covered by scales in T. longipectoralis Zheng, Du, Chen & Yang, 2009 and T. yunnanensis); lateral line complete (vs incomplete in T. huanjiangensis); and pelvic-fin tip not reaching to anus (vs reaching to anus in T. gejiuensis, T. macrocephala Yang, Wu & Yang, 2012, T. rosa and T. qiubeiensis).

Description

Morphometric data of the type specimens of T. wulongensis are presented in Table 3. D, 2/8–9; A, 1/5–6; P, 1/8–9; V, 1/5–7; C, 18; vertebrae: 4+38–39 (five specimens).

Table 3.

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Morphometric data of type specimens of Triplophysa wulongensis sp. nov. SD = standard deviation.

Morphometric characters	Holotype	Paratypes (SWU2019051301–08)
Morphometric characters	SWU2019051309	Min	Max	Mean	SD
SL (mm)	64	49	67.2	55.7
% SL
Lateral head length (HL)	22.9	20.4	23.5	22.6	1
Body depth	13.3	9.3	13.6	12.1	1.3
Predorsal length	54.2	50.4	53.3	51.9	0.9
Postdorsal length	40.7	34	39.4	36.7	1.4
Prepelvic length	49.6	48.3	50.9	49.7	0.9
Preanal length	76	71.5	77.7	73.4	1.8
Preanus length	70	67.4	70.2	69	1
Dorsal-fin height	15.9	15	19.8	16.5	1.5
Dorsal-fin base length	12	10.7	13.4	12.4	0.9
Anal-fin height	14.1	12.4	16.5	14.5	1.3
Anal-fin base length	6.6	6.6	8.2	7.4	0.6
Pelvic-fin length	12.4	12.5	14.5	13.2	0.6
Pectoral-fin length	16.6	15.6	18.4	17.6	1
Caudal-fin length	17.8	15.9	20.8	18.1	1.3
Caudal-peduncle length (CPL)	20.8	14.2	18.4	16.6	1.2
Caudal-peduncle depth (CPD)	9.4	7.6	9.4	8.5	0.7
Pectoral-pelvic distance	26.8	24.6	28.6	26.6	1.1
Pelvic-anal distance	26.4	21.6	26.9	23.7	1.4
Vent-anal fin origin distance	6.2	4	7.2	5.8	1
%HL
Head depth	53.8	45.3	54.2	50.6	2.9
Head width	62.2	55.7	65.8	62.4	3.5
Snout length	39	38.9	45	41.9	1.9
Eye diameter	11.1	12.2	19.1	17	2.1
Interorbital width	38.7	38.5	43.1	41.3	1.5
Postorbital head length	45	37.9	46.8	43.8	2.8
Maxillary barbel length	21.8	27.2	35.9	29.8	3.2
Inner rostral barbel length	16.5	20.1	23.4	21.6	1.2
Outer rostral barbel length	21.4	25.9	41.5	32.4	4.5
CPD/CPL	45.3	44.3	57.4	51.2	4.5

Body elongated, slightly compressed anteriorly and more strongly compressed posteriorly. Deepest point of body in front of dorsal fin origin, body depth 9.3–13.6% of SL. Caudal peduncle depth/caudal peduncle length range from 44.3% to 57.4%. Head depressed, width greater than depth (62.4% vs 50.6% of HL). Snout moderately blunt and snout length almost equal to postorbital length, approximately 38.9–45.0% of HL. Anterior and posterior nostrils adjacently located; anterior nostril in short tube, each with tip elongated to form a short barbel. Tip of nostril appendage not reaching the anterior margin of eyes. Eyes present, diameter 11.1–19.1% of HL. Mouth inferior, arched; mouth corner situated below anterior nostril. Lips thin; lower lip with well-marked, V-shaped, median notch (Fig. 2). Upper jaw covered by upper lip; lower jaw scoop-shaped, not covered medially by lower lip. Three pairs of barbels; inner rostral barbel extending to rictus, 16.5–23.4% of HL; outer rostral barbel not extending to anterior margin of eyes, 21.4–41.5% of HL; maxillary barbel extending to anterior margin of eyes, 21.8–35.9% of HL.

Figure 2.

Triplophysa wulongensis sp. nov., holotype SWU 2019051309, 64.0 mm SL; head in dorsal and ventral view.

Dorsal fin emarginate, origin posterior to pelvic fin insertion, situated slightly posterior to midpoint between snout tip and caudal fin base; first branched ray longest; dorsal fin height shorter than lateral head length; tip of dorsal fin reaching vertical of anus. Pectoral fin moderately developed, 56.6–72.9% of distance between pectoral fin and pelvic-fin origins. Pelvic-fin origin situated almost at midpoint between pectoral-fin origin and anal-fin origin, tip of pelvic fin not reaching to anus. Anal-fin origin situated almost at midpoint between pelvic-fin origin and caudal-fin base, distal margin of anal fin truncate; posterior tip of anal fin reaching approximately half distance between anal-fin origin and caudal-fin base. Vent-anal fin-origin distance 4.0–7.2% of SL. Caudal fin emarginate.

Body smooth and scaleless. Cephalic lateral line system developed. Lateral line complete, ending at caudal-fin base. Intestine without bends or loops immediately posterior to stomach; stomach U-shaped. Posterior chamber of gas bladder degenerate.

Coloration

In formalin-fixed specimens, body yellowish dorsally, gradually lighter toward ventral side. Fins semitransparent. Body dorsally and laterally covered with irregular, brown blotches; 6–8 distinct dark brown blotches along dorsal midline.

Sexual dimorphism

Sexual dimorphism was not detected. This may reflect that the sampling time was outside the breeding season of this species.

Geographical distribution

Known only from the type series, from a pool in Furong Cave, connected to the Wujiang River near Wulong, (Fig. 3). Triplophysa wulongensis was found syntopic with T. rosa.

Figure 3.

Collection site of Triplophysa wulongensis sp. nov. (red triangle) in Chongqing, Southwest China.

Etymology

The specific name, wulongensis, refers to the type locality in Wulong County, where the type specimens were collected; it is an adjective with alternative endings -is and -e.

Discussion

In previous studies, the cave dwelling species of Triplophysa were nested in a basal position to congeners in phylogenetic reconstructions (Wang et al. 2016; Chen and Peng 2019; Wu et al. 2020). Our phylogenetic analysis based on cytb (Fig. 4) resolved two monophyletic clades, one of which comprises cave-dwelling species, and the other includes non-cave-dwelling species, concordant with Chen and Peng (2019). Triplophysa wulongensis is located in a basal position of the cave-dwelling clade (Fig. 4). The K2P genetic distances show less differentiation between T. wulongensis and T. sanduensis (14.9%) than between T. wulongensis and its other congeners in this study. The K2P genetic distance (ranges from 14.9% to 24.9%) between the new species and some of the other species of Triplophysa based on cytb markers is consistent with species-level divergences in other fish taxa (Ward et al. 2005; Wang et al. 2016; Wu et al. 2020).

Figure 4.

Phylogeny of some species of Triplophysa and two outgroup species based on maximum likelihood (ML) and Bayesian inference (BI) methods using mitochondrial cytb gene sequences. The ML bootstrap values and BI posterior probabilities are shown at the nodes

The presence or absence of the secondary sexual characteristics is important for the generic diagnosis of loaches (Bănărescu and Nalbant 1968; Zhu 1989). The presence of tubercles on the pectoral fin can be considered as an autapomorphy and is the single diagnostic character of Triplophysa (Prokofiev 2010). Nevertheless, according to Liang and Zhou (2019), some cave-dwelling species of Triplophysa, e.g. T. nasobarbatula and T. zhenfengensis, have lost secondary sexual characteristics. Sexual dimorphism was not evident in the type series of T. wulongensis, but the phylogenetic analysis confirmed the generic classification.

The majority of the cave-dwelling species of Triplophysa were described from karst caves and subterranean streams in the Pearl river basin and the upper Yangtze river basin, with an additional two species (T. erythraea and T. xiangxiensis) reported from the Yuanjiang river drainage (a tributary of the middle Yangtze River) and a single species (T. rosa) described from the Wujiang river drainage (Lan et al. 2013; Liu et al. 2017; Wu et al. 2018b; Chen and Peng 2019; Huang et al. 2019). In terms of morphology, T. wulongensis is distinguished from the co-occurring T. rosa by the presence of eyes (vs absence), 8 or 9 branched pectoral-fin rays (vs 12), 18 branched caudal-fin rays (vs 14), a pectoral fin length that is 15.6–18.4% that of the SL (vs 26.6%), and a body with irregular brown blotches (vs pale blotches).

The rate of discovery of new cave-dwelling species of Triplophysa has increased in recent years (Yang et al. 2016; Li et al. 2017a, b, 2018; Liu et al. 2017; Wu et al. 2018a, b; Chen and Peng 2019; Huang et al. 2019), while a taxonomic revision of these species is lacking. Hence, further systematic and phylogenetic study based on both morphometric and molecular approaches is needed.

Key to the cave-dwelling species of Triplophysa

1	Eyes normal	2
–	Eyes reduced or absent	16
2	Scales absent	3
–	Body covered by scales	8
3	Tip of pelvic fin reaching anus, outer gill rakers on first gill arch absent	*T. longliensis*
–	Tip of pelvic fin not reaching anus; outer gill rakers on first gill arch present	4
4	Posterior chamber of air bladder developed; anterior nostril not elongate to barbel-like	*T. tianxingensis*
–	Posterior chamber of air bladder degenerated; anterior nostril elongate to barbel-like	5
5	Dorsal-fin origin closer to caudal-fin base than to snout tip	T. wulongensis sp. nov.
–	Dorsal-fin origin closer to snout tip than to caudal-fin base	6
6	Dorsal-fin origin opposite vertical line trough pelvic-fin origin	*T. nanpanjiangensis*
–	Dorsal-fin origin anterior to vertical line trough pelvic fin origin	7
7	Caudal fin deep forked with 11–13 branched fin rays	*T. baotianensis*
–	Caudal fin slightly forked with 14 branched fin rays	*T. xiangshuingensis*
8	Processus dentiformis present in upper jaw	*T. zhenfengensis*
–	Processus dentiformis absent in upper jaw	9
9	Posterior chamber of air bladder developed	*T. guizhouensis*
–	Posterior chamber of air bladder degenerated	10
10	Tip of depressed pelvic fin exceeding anus	11
–	Tip of depressed pelvic fin not reaching anus	13
11	Anterior nostril not elongate to barbel-like; branched dorsal-fin rays 10	*T. flavicorpus*
–	Anterior nostril elongate to barbel-like; branched dorsal-fin rays 8	12
12	Tip of pectoral fin extending beyond pelvic-fin origin	*T. longipectoralis*
–	Tip of pectoral fin not reaching pelvic-fin origin	*T. nasobarbatula*
13	Branched dorsal-fin rays 7, branched anal-fin rays 7	*T. yunnanensis*
–	Branched dorsal-fin rays 8, branched anal-fin rays 5–6	14
14	Dorsal-fin origin opposite vertical line trough pelvic-fin origin	*T. nandanensis*
–	Dorsal fin origin anterior to vertical line trough pelvic fin origin	15
15	Anterior nostril not elongate to barbel-like; branched caudal-fin rays 16	*T. huapingensis*
–	Anterior nostril elongate to barbel-like; branched dorsal-fin rays 17–18	*T. sanduensis*
16	Eyes reduced	17
–	Eyes absent	24
17	Body covered with scales	18
–	Scales absent, body smooth	19
18	Lateral line complete, branched anal-fin rays 6	*T. luochengensis*
–	Lateral line incomplete, branched anal-fin rays 5	*T. lingyunensis*
19	Lateral line incomplete; adipose keels present on upper or lower side of caudal peduncle	*T. langpingensis*
–	Lateral line complete; adipose keels absent from caudal peduncle	20
20	Posterior chamber of air bladder developed	*T. xichouensis*
–	Posterior chamber of air bladder degenerated	21
21	Tip of pelvic fin reaching anus	22
–	Tip of pelvic fin not reaching to anus	23
22	Tip of pectoral fin reaching to midway between pectoral-fin origin and pelvic-fin origin; Spots absent from body	*T. tianlinensis*
–	Tip of pectoral fin reaching a vertical through dorsal-fin origin; spots present on body	*T. macrocephala*
23	Dorsal-fin origin posterior to or at to vertical line trough pelvic-fin origin; branched caudal-fin rays 13	*T. aluensis*
–	Dorsal-fin origin anterior to vertical line trough pelvic-fin origin; branched caudal-fin rays 15–16	*T. tianeensis*
24	Lateral line absent	*T. huanjiangensis*
–	Lateral line complete	25
25	Tip of pelvic-fin not reaching to anus	26
–	Tip of pelvic fin reaching to anus	28
26	Adipose keels present on upper or lower side of caudal peduncle	*T. posterodorsalus*
–	Adipose keels absent from caudal peduncle	27
27	Branched dorsal-fin rays 8; branched caudal-fin rays 16	*T. fengshanensis*
–	Branched dorsal-fin rays 7; branched caudal-fin rays 14	*T. shilinensis*
28	Anterior nostril not elongate to barbel-like	29
–	Anterior nostril elongate to barbel-like	31
29	Lips developed, papillary process absent, branched caudal-fin rays 17	*T. erythraea*
–	Lips developed, papillary process present, branched caudal-fin rays 14–15	30
30	Branched dorsal fin rays 8; branched pectoral-fin rays 11	*T. maolanensis*
–	Branched dorsal-fin rays 7; branched pectoral-fin rays 7–9	*T. qiubeiensis*
31	Distal margin of dorsal fin truncate; branched dorsal-fin rays 7–8; branched pectoral-fin rays 9–11; branched pelvic-fin rays 6	32
–	Distal margin of dorsal-fin concave; branched dorsal-fin rays 9; branched pectoral-fin rays 12; branched pelvic-fin rays 7	*T. rosa*
32	Snout blunt; tip of pectoral fin not reaching vertical level of dorsal fin origin; tip of caudal-fin lobes pointed; branched caudal-fin rays 14–15	33
–	Snout rectangle-like; tip of pectoral fin reaching a vertical through dorsal-fin origin; tip of caudal-fin lobe sharp; branched caudal-fin rays 16	*T. xiangxiensis*
33	Cephalic lateral-line canals with 5 supraorbital and 7 preoperculo-mandibular pores	*T. gejiuensis*
–	Cephalic lateral-line canals with 8 supraorbital and 12–13 preoperculo-mandibular pores	*T. anshuiensis*

Acknowledgements

We are indebted to Ms Ni Liu for her help with the specimen collections. This work was supported by grant from the National Natural Science Foundation of China (31872204).

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Supplementary material

Supplementary material 1

Table S1

Shijing Chen, Bakhtiyor Sheraliev, Lu Shu, Zuogang Peng

Data type: Specimen list

Explanation note: Material examined of Triplophysa species from China.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

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