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

Abstract We describe a new species of cave-dwelling loach, Triplophysa wulongensissp. 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.


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).
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.

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).
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 pectoralfin 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.
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. 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 (  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 Li et al. 2017aLi et al. , b, 2018Liu 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