A new species of Tibetan loach, Triplophysa weiheensis sp. nov., is described from the Weihe River in Gansu Province, China, based on morphological and molecular analyses. The new species can be distinguished from all known congeners by a unique combination of the following characters: scaleless; snout abruptly sloping downward, anterior to anterior nostril; lower jaw crescentic, not sharp; body without obvious mottling; lateral line interrupted on posterior trunk at pelvic-fin distal extremity; caudal-peduncle length 2.0–2.7 times its depth; branched rays of pectoral fin 10–11; branched rays of pelvic fin 5–6; inner gill rakers on 1st gill arch 14–16; vertebrae 4+34–36; intestine with 6–7 loops, length ca. 1.8 times SL (n=3); bony capsule of air bladder small and thin; posterior chamber of air bladder absent.
Species of the genus Triplophysa Rendahl 1933 are the most common fish among the nemacheilids found in the Qinghai-Tibet Plateau (QTP) (Wu & Wu, 1992; Zhu, 1989). These fish are found in almost all water bodies in the region, with new species of Triplophysa still being reported (Huang et al., 2019; Liu et al., 2017; Wu et al., 2018; Yang et al., 2016). As a result, a total of 147 valid species of Triplophysa have been recorded to date (Froese & Pauly, 2019).
Weihe River is a tributary of the Yellow River and originates from the southern part of Gansu Province (Figure 1A). Previous studies have reported that southern Gansu is a hotspot area for Triplophysa, with an extensive distribution of species across the river systems (Feng et al., 2017a, 2017b, 2019b). Until now, 17 species of Triplophysa have been reported from the Yellow River system (Chen et al., 1987; Ding, 1994; Feng et al., 2017a; Wu & Wu, 1992; Zhu, 1989), five of which have been recorded from Weihe River (Supplementary Table S1). Following an investigation of Triplophysa species from Weihe River (Figure 1A), 15 specimens superficially resembling Triplophysa stoliczkae Steindachner 1866 (Supplementary Figure S1) were collected and are described herein as a new species based on morphological and molecular analyses.
Figure 1. Collection site, morphological characters and phylogenetic position of Triplophysa weiheensis sp. nov.
After euthanization (see Supplementary Methods), the left ventral fin of some specimens was removed and preserved in 95% ethanol for DNA extraction. Voucher specimens were labeled and stored in 70% ethanol. Specimens were deposited in the collection of the Northwest Institute of Plateau Biology (NWIPB), Chinese Academy of Sciences, Xining, Qinghai, China. Morphological measurements and counts followed Kottelat (1990) and Prokofiev (2007). Additional measurements are described in the Supplementary Methods. Measurements were taken with digital calipers to the nearest 0.1 mm. Previous research has reported that T. stoliczkae is a striking case of morphological convergence and consists of distinct lineages that are not close relatives (Feng et al., 2019a). As there is no formal taxonomic revision for T. stoliczkae, we treated it as a morphological species in this study but considered its different genetic lineages in phylogeny. These lineages, which were initially mistaken as T. stoliczkae, exhibit very similar morphology. Thus, they represent a known morphological unit in the genus Triplophysa. We specifically measured 61 T. stoliczkae specimens collected from various water systems and used principal component analysis (PCA) (Supplementary Table S2) to visualize morphological differences between T. stoliczkae and the new species. Furthermore, we employed a Micro CT (Quantum GX2, PerkinElmer Corporation, USA) to build a skeletal model of the new species.
DNA extraction and complete cyt b gene (1140 bp) amplification were carried out, as detailed in the Supplementary Methods. Polymerase chain reaction (PCR) products were sequenced from both directions using an ABI PRISM 3700 sequencing system. Finally, 20 new cyt b sequences of Triplophysa were obtained, including five sequences from the new species (Supplementary Table S3). To assess the phylogenetic status of the new species, 37 additional cyt b sequences of Triplophysa and outgroups (Barbatula toni and Lefua costata) were retrieved from GenBank (Supplementary Table S3), as per previous studies (Feng et al., 2017b; He et al., 2006; Wang et al., 2016). Phylogenetic analysis was performed using Bayesian inference (BI) (Supplementary Methods). Additionally, Kimura’s 2-parameter (K2P) genetic distances (Kimura, 1980) were estimated using MEGA 6.0 (Tamura et al., 2013).
Morphometric character Holotype Paratype (Range n=14) Mean SD Standard length (mm) 65.8 48.3–77.1 Percentage of standard length (%) Body depth 18.1 16.2–19.2 18.2 0.9 Body width 16.5 14.1–17.6 16.0 1.0 Head length 17.9 16.3–21.5 19.6 1.7 Dorsal-fin length 16.9 14.1–20.6 17.7 1.9 Pelvic-fin length 14.2 12.2–14.7 13.6 0.7 Pectoral-fin length 18.2 15.1–20.5 17.3 1.8 Anal-fin length 15.2 13.1–17.1 15.0 1.2 Caudal-fin length 21.1 16.4–22.4 19.3 1.6 Predorsal length 49.5 47.9–55.1 50.9 2.2 Preanus length 68.5 61.9–71.0 67.3 2.6 Preanal length 72.2 63.5–73.4 69.8 2.5 Prepelvic length 56.7 51.1–58.4 54.6 2.4 Prepectoral length 19.7 16.9–21.9 19.8 1.3 Caudal-peduncle length 23.1 19.4–24.3 22.0 1.3 Caudal-peduncle depth 10.3 8.7–10.7 9.7 0.6 Pectoral-pelvic distance 32.5 31.6–38.8 35.2 2.4 Pectoral-anal distance 48.4 46.1–54.9 50.3 2.9 Pelvic-anal distance 15.5 12.7–18.1 15.3 1.7 Head length (mm) 11.8 10.0–14.0 Percentage of head length (%) Head depth 67.4 59.2–72.2 63.5 4.4 Head width 81.9 68.1–91.3 79.7 8.0 Snout length 35.6 31.2–38.8 35.0 2.3 Eye length 19.4 16.6–22.9 18.7 1.7 Interorbital width 34.7 28.6–43.0 36.2 4.0 Postorbital length 50.6 41.4–53.0 48.4 3.1 Inner rostral barbel length 28.9 15.1–28.1 21.6 3.6 Outer rostral barbel length 27.0 16.7–27.5 22.9 2.9 Maxillary barbel length 31.4 18.7–28.9 23.4 3.2
Table 1. Morphometric data of Triplophysa weiheensis sp. nov.
Holotype: NWIPB 1505189, 65.8 mm SL (standard length); Zhang River, a tributary of Weihe River, at Chenjiamo Village, Zhangxian County, Dingxi City, Gansu Province, China (N34°48′, E104°31′; elevation 1 768 m a.s.l.), collected by Chenguang Feng, Chao Tong, and Kai Zhao on 12 May 2015.
Paratypes: NWIPB 1505181–183, 3 ex. (examined specimens), 64.1–77.1 mm SL; 1505185–188, 4 ex., 59.7–68.0 mm SL, same collection information as holotype. NWIPB 1505922, 53.6 mm SL, Niutou River, a tributary of Weihe River, at Maiji District, Tianshui City, Gansu Province, China (N34°34′, E105°57′; elevation 1 360 m a.s.l.), on 21 May 2015. NWIPB 1505985–988, 4 ex., 56.4–65.9 mm SL; 1505990, 48.3 mm SL; 1505992, 60.5 mm SL, Niutou River, a tributary of Weihe River, at Hongbao Town, Qingshui County, Tianshui City, Gansu Province, China (N34°42′, E106°1′; elevation 1 434 m a.s.l.), on 22 May 2015.
Etymology: The specific epithet weiheensis is derived from Weihe River (渭河 in Chinese, type locality) with the Latin suffix -ensis.
Diagnosis: Triplophysa weiheensis sp. nov. can be distinguished from all known congeners by a combination of the following characters: (1) scaleless; (2) snout abruptly bending down before anterior nostril; (3) snout length shorter than postorbital length; (4) lower jaw crescentic, not sharp; (5) body without obvious mottling; (6) lateral line interrupted on posterior trunk behind vertical line of pelvic-fin distal extremity; (7) caudal-peduncle length 2.0–2.7 times its depth; (8) caudal fin slightly emarginate; (9) branched rays of pectoral fin 10–11; (10) pelvic-fin insertion behind vertical line through dorsal-fin origin, distal fin tip attaining anal-fin origin when adpressed, branched rays 5–6; (11) inner gill rakers on 1st gill arch 14–16; (12) vertebrae 4+34–36; (13) intestine with 6–7 loops, length ca. 1.8 times SL; (14) posterior chamber of air bladder absent, bony capsule of air bladder small and thin.
Description: Morphometric and meristic data are given in Table 1 and Supplementary Table S4, respectively.
Body thick, cylindrical. Dorsal profile of body arch-like (Figure 1B). Maximum depth of body slightly greater than maximum width, occurring between pectoral and dorsal fins. Caudal peduncle laterally compressed, depth nearly uniform toward caudal-fin base, length longer than head length (HL, 101.0–133.7% of HL). Head width greater than depth. Cheeks slightly inflated, V-shaped outline in ventral view. Snout obtuse, sloping downward anterior to anterior nostril (Figure 1B, C). Snout length shorter than postorbital length. Anterior and posterior nostrils close together. Valves around anterior nostrils, but not around posterior. Eyes small, dorsolaterally in head. Interorbital space wide (28.6–43.0% of HL, 155.6–239.0% of eye length). Mouth inferior (Supplementary Figure S2). Lips thick and well-developed with furrows; lower lip continuous with shallow median incision. Lower jaw crescentic, uncovered by lower lip. Three pairs of barbels thick, moderately short; inner rostral barbels almost reaching corner of mouth; outer rostral barbels horizontally reaching posterior nostril; maxillary barbels horizontally reaching mid-point of eyes.
Fins short (Figure 1B). Dorsal fin rounded distally, originating anterior to pelvic-fin origin; dorsal-fin origin near midway between tip of snout and caudal-fin base or slightly nearer to caudal-fin base. Pectoral fin short, not reaching halfway point to pelvic-fin base. Pelvic fin reaching past anus, distal fin tip attaining anal-fin origin when adpressed. Anal fin rounded distally, just posterior to anus. Caudal fin slightly emarginate.
Skin scaleless. Lateral line tapering, interrupted on posterior trunk at pelvic-fin distal extremity; few lateral line pores close to caudal-fin base. Stomach U-shaped, expanded. Intestine long, with 6–7 loops (Figure 1D and Supplementary Figure S3; one hidden loop surrounded by 5–6 loops). Bony capsule of air bladder small and thin, closed laterally; posterior chamber of air bladder absent (Figure 1E).
Sexual dimorphism: In mature males, unbranched and four outer branched pectoral-fin rays thickened, covered with breeding tubercles on dorsal surface. Small breeding tubercles also present on both sides of head in liber apophyses extending from anterior lower margin of orbit to base of outer rostral barbel. These characters do not occur in females (Figure 1F).
Color pattern: In life: silver-gray base in individuals. In 70% EtOH: ground color of body brown dorsally and laterally, becoming yellowish ventrally. Fin membranes hyaline and slightly gray, with dark-brown faint spots on both sides of dorsal and caudal fins and upper side of pectoral fin. Body without obvious mottling. Peritoneum silvery with scattered dark melanophores becoming dense at vertebral column.
Ecology: Specimens were collected from flowing streams with gravel or sandy substrates at 1 360–1 768 m a.s.l. (Supplementary Figure S4). River water became muddy at about 0.5 m deep. Periphytic algae, sand, and insect larvae (e.g., chironomids) were found in the stomach of specimens. Other species collected with Triplophysa weiheensis sp. nov. included T. dalaica Kessler, T. minxianensis Wang and Zhu, Pseudorasbora parva Temminck and Schlegel, and Gobio huanghensis Lo, Yao et Chen.
Distribution: Triplophysa weiheensis sp. nov. is known only from the upper reaches of the Weihe River (Figure 1A).
Comparisons: Intestine and posterior chamber of air bladder are two important characters in the morphological taxonomy of the genus Triplophysa (He et al., 2006; Wu and Wu, 1992; Zhu, 1989). Triplophysa weiheensis sp. nov. differs from most Triplophysa species based on the combination of intestine screw shaped and posterior chamber of air bladder absent (Figure 1D, E), and can be classified into the same category as T. stoliczkae Steindachner, T. tanggulaensis Zhu, T. crassilabris Ding, T. alticeps Herzenstein, T. cakaensis Cao and Zhu, T. chondrostoma Herzenstein, T. stenura Herzenstein, T. rotundiventris Wu and Chen, T. nujiangensa Chen et al., and T. daochengensis Wu et al. (Chen et al., 2004; Ding, 1994; Wu & Wu, 1992; Wu et al., 2016; Zhu, 1989). Among these, the new species is most similar to T. stoliczkae.
Triplophysa weiheensis sp. nov. can be distinguished from T. stoliczkae by a combination of the following characters: lower jaw crescentic, not sharp (vs. spade-like, sharp); lateral line incomplete (vs. complete); vertebrae 4+34–36 (vs. 4+38–41); intestine ca. 1.8 times as long as SL (vs. 1.0–1.3 times); body without obvious mottling (vs. with mottling). The PCA results also indicated that the new species differs from T. stoliczkae in its whole morphology (Figure 1G) and the two species can be clearly separated from each other by PC1. Five morphometric measurements, including head depth/head length, interorbital width/head length, body depth/standard length, caudal peduncle depth/standard length, and postorbital length/head length, were highly correlated with and substantially contributed to PC1 (Supplementary Figure S5A, B). The five morphometric measurements were greater in the new species than in T. stoliczkae (Supplementary Figure S5C), suggesting a comparatively stubby body for the new species relative to T. stoliczkae.
Additionally, Triplophysa weiheensis sp. nov. can be distinguished from T. tanggulaensis by the following characters: pelvic fin i, 5–6 (vs. i, 7–9); inner gill rakers on 1st gill arch 14–16 (vs. 10–13); vertebrae: 4+34–36 (vs. 4+37–38); intestine with 6–7 loops (vs. 3–4 loops). The new species can be distinguished from T. crassilabris by the following characters: pectoral fin i, 10–11 (vs. i, 8–9); pelvic fin i, 5–6 (vs. i, 7–8); inner gill rakers on 1st gill arch 14–16 (vs. 8–9); intestine with 6–7 loops (vs. 4–5 loops). The new species can be distinguished from T. alticeps by the following characters: intestine with 6–7 loops (vs. 3–4 loops); bony capsule of air bladder small and thin (vs. large and inflated). The new species can be distinguished from T. cakaensis by the following characters: snout abruptly sloping downward anterior to nostril (vs. snout gently sloping downward); lateral line interrupted on posterior trunk at pelvic fin distal extremity (vs. ending above pectoral fin); body without obvious mottling (vs. with mottling). The new species can be distinguished from T. chondrostoma by the following characters: lower jaw crescentic, not sharp (vs. spade-like, sharp); bony capsule of air bladder small and thin (vs. large and inflated); caudal-peduncle length 2.0–2.7 times its depth (vs. 3.2–3.8 times); body without obvious mottling (vs. with distinct mottling). The new species can be distinguished from T. stenura by the following characters: caudal peduncle depth nearly uniform towards caudal-fin base (vs. tapered); lateral line interrupted (vs. complete); intestine ca. 1.8 times as long as SL (vs. 1.0–1.3 times). The new species can be distinguished from T. rotundiventris by the following characters: caudal-peduncle length 2.0–2.7 times its depth (vs. 3.0–3.6 times); intestine ca. 1.8 times as long as SL (vs. 2.0–2.5 times); body without obvious mottling (vs. with mottling). The new species can be distinguished from T. nujiangensa by the following characters: vertebrae: 4+34–36 (vs. 4+38–39); intestine with 6–7 loops (vs. 3 loops); pelvic fin reaching past anus (vs. not reaching). The new species can be distinguished from T. daochengensis by the following characters: intestine with 6–7 loops (vs. 3 loops); pelvic fin reaching past anus (vs. not reaching); caudal fin slightly emarginate (vs. deeply emarginate).
Molecular analysis: Our results were in accordance with previous study, which suggested that T. stoliczkae consists of distinct lineages that are not close relatives (Feng et al., 2019a; Figure 1H). Phylogenetic analysis recovered the monophyly of Triplophysa weiheensis sp. nov. with strong support and showed that it was close to the T. stoliczkae population from the Yellow River system. The K2P genetic distance between Triplophysa weiheensis sp. nov. and T. stoliczkae was 3.7%, which is larger than that between several pairs of recognized species (Supplementary Table S5). These analyses suggest that Triplophysa weiheensis sp. nov. is a separately evolving lineage and genetic differences from its sister lineage in the genus Triplophysa may have reached species-level differentiation. Morphologically, this distinct phylogenetic lineage differed from all described Triplophysa species. Thus, based on an integrative taxonomic approach (Wu et al., 2019), the specimens collected from Weihe River are designated as a distinct species.