Troglonectes canlinensis sp. nov. (Teleostei: Nemacheilidae), a New Troglomorphic Loach from Guangxi, China

Simple Summary Troglonectes is a small-body loach endemic to the Guangxi and Guizhou provinces of China, showing a particular affinity for cave areas. Twenty species were recorded in this genus, including one new species. The new species, Tr. canlinensis, can be distinguished from other congenetic species by their morphological characteristics and molecular evidence. In the genus of Troglonectes, the eye, lateral line and scale present or absent, the number of branched pectoral fin rays, caudal fin rays and anal fin rays, and the depth of the upper adipose keel on the caudal peduncle are important identifying characteristics. Abstract A new species of the genus Troglonectes is described based on specimens from a karst cave in Andong Town, Xincheng County, Liuzhou City, Guangxi, China. Troglonectes canlinensis sp. nov. can be distinguished from its congener species by the following combination of characteristics: eye degenerated into a black spot; whole body covered by scales, except for the head, throat, and abdomen; incomplete lateral line; forked caudal fin; 8–10 gill rakers on the first gill arch; 13–14 branched caudal fin rays; 8–9 branched dorsal fin rays; 5–6 anal fin rays; 9–10 pectoral fin rays; upper adipose keel depth mostly 1/2 of the caudal peduncle depth; and caudal fin forked.


Introduction
Cave loaches of the genus Troglonectes Zhang, Zhao, and Yang, 2016 (abbreviation is Tr. in this study in order to differ from the abbreviation of Triplophysa) are small-bodied fish that mainly occur in the Guangxi and Guizhou provinces of China, showing a particular affinity for cave areas. Troglonectes was separated from the genus Oreonectes Günther, 1838, that Du et al. [1] divided Oreonectes into the platycephalus group, i.e., caudal fin rounded or truncated, and the furcocaudalis group, i.e., caudal fin forked. Subsequently, Zhang et al. [2] proposed the genus Troglonectes and assigned seven nominal species to Troglonectes, i.e., Tr. acridorsalis (Lan, 2013), Tr. barbatus (Gan, 2013), Tr. elongatus (Tang, Zhao, and Zhang, 2013), Tr. macrolepis (Huang, Du, Chen, and Yang, 2009), Tr. microphthalmus (Du, Chen, and Yang, 2008), and Tr. translucens (Zhang, Zhao, and Zhang, 2006), in addition to the type species Tr. furcocaudalis (Zhu and Cao, 1987). Troglonectes can be distinguished from other genera in the Nemacheilidae by possessing narrowly separated nostrils, tube-shaped anterior nostril, tip of the anterior nostril extending into the barbel, dorsal fin origin anterior to the pelvic fin origin, and caudal fin forked or truncated [2]. Except for the species of Oreonectes placed in Troglonectes, some species of Paracobitis and Triplophysa were also placed in Troglonectes based on their morphology and molecular evidence. Chen et al. [3] described P. longibarbatus Chen, Yang, Sket, and Aljancic, 1998 from Libo County, Guizhou, but Du et al. [1] placed it in the Triplophysa, based on the morphological characteristics, elongated barbel-like anterior nostril, and sexual dimorphism present in males. Li et al. [4] and Lin et al. [5] described P. maolanensis Li, Ran, and Chen, 2006 and T. jiarongensis Lin, Li, and Song, 2012 from Guizhou Province, respectively. However, Huang et al. [6] and Luo et al. [7] placed P. longibarbatus, P. maolanensis, and T. jiarongensis in the Troglonectes. Subsequently, Luo et al. [7] treated T. jiarongensis as a synonym of Tr. elongatus. Additionally, Huang et al. [6] mentioned that O. daqikongensis Deng, Wen, Xiao, and Zhou, 2016 and O. shuilongensis Deng, Wen, Xiao, and Zhou, 2016 also belong to the genus Troglonectes due to the forked caudal fin, dorsal fin originating anterior to the pelvic fin origin, and presence of caudal crests. Luo et al. [7] placed T. huanjiangensis Yang, Wu, and Lan, 2011, T. lihuensis Wu, Yang, and Lan, 2012, T. lingyunensis (Liao, Wang, and Luo, 1997), and O. retrodorsalis Lan, Yang, and Chen, 1995 in the Troglonectes based on molecular analysis. Zhao et al. [8] described one new species, T. hechiensis Zhao, Liu, Du, and Luo, 2021, and stated that 17 species were contained within the Troglonectes. Luo et al. [7]  In July 2022, 10 specimens of Troglonectes were collected from a cave in Andong Town, Xincheng County, Liuzhou City, Guangxi Zhuang Autonomous Region (hereinafter referred to as Guangxi), China. Morphological and molecular evidence supported these loach specimens representing a new species of Troglonectes. Hence, the new species is described herein.

Materials and Methods
All care and use of experimental animals complied with the relevant laws of the Chinese Laboratory of Animal Welfare and Ethics (GB/T 35892-2018). Specimens of Troglonectes canlinensis sp. nov. were collected by F.G. Luo and euthanized rapidly by an overdose of clove oil anesthetic. The right-side pectoral fin and pelvic fin of one specimen were removed and preserved in 99% ethanol. The specimens for the morphological study were stored in 10% formalin, then transferred to 75% alcohol for long-term preservation in the Kunming Natural History Museum of Zoology, Kunming Institute of Zoology (KIZ), Chinese Academy of Science (CAS), China.
Counts and measurements followed Du et al. [1,9], Tang et al. [10], and Lan et al. [11]. Complete mitochondrial genes were sequenced by the Science Corporation of Gene (China) following standard Illumina protocols. Genome sequencing data were submitted to Gen-Bank under Accession No. OQ129618. We retrieved twenty-one complete mitochondrial genomes and five cytb reference sequences of twenty-four Nemacheilidae and two Botiidae species from the NCBI GenBank database for phylogenetic tree reconstruction. Parabotia fasciata Dabry de Thiersant, 1872 and Leptobotia elongata (Bleeker, 1870), two species of Botiidae, were used as outgroups. To test the phylogenetic position of Troglonectes canlinensis sp. nov., Bayesian inference (BI) analysis was performed using MrBayes on XSEDE (v3.2.7a) and the CIPRES Science Gateway [12]. Two runs were performed simultaneously with four Markov chains starting from a random tree. The chains were run for five million generations and sampled every 100 generations. The first 25% of the sampled trees were discarded as burn-in, and the remaining trees were used to create a consensus tree and estimate the Bayesian posterior probabilities (BPPs). from T. retrodorsalis by the tip of the anterior nostril being elongated to barbel-like and the nostril barbel length being nearly twice the nostril tube length (vs. nostril barbel length being less than 1/2 of the tube length).

Troglonectes
Description. The morphometric data of the type specimens of Troglonectes canlinensis sp. nov. are given in Table 1. Dorsal fin with 4 unbranched and 8-9 branched rays; anal fin with 3 unbranched and 5-6 branched rays; pectoral fin with 1 unbranched and 9-10 branched rays; pelvic fin with 1 unbranched and 5-6 branched rays, caudal fin with 13-14 branched rays; and 8-10 inner-gill rakers on the first gill arch. Vertebrae 4 + 34 (one specimen) Body elongated, slightly flattened in front, strongly compressed in back. Dorsal profile convex and ventral profile straight in live specimen, but it inversed in preserved specimens. From snout to dorsal fin origin, the body depth increases to its maximum, maximum body depth of 18.2-21.3% SL. Head slightly depressed and flattened, maximum head width greater than the deepest head depth. Anterior and posterior nostrils adjacent, distance less than the diameter of the posterior nostril. Eyes reduced, eye diameter 7.5-11.6% of the lateral head length. Mouth inferior, snout obtuse, upper and lower lips with small furrows and without papillae, median of the lower lip with a V-shaped notch. Three pairs of barbels, inner, outer, and maxillary barbels, extend vertically to the posterior margin of the anterior nostril, anterior margin of the eye, and preopercle, respectively.   Distal margin of dorsal fin truncates, origin anterior to the pelvic fin origin, predorsal length of 54.2-58.6% SL. Tip of pectoral fin reaching halfway to the pelvic fin origin. Tip of pelvic fin far away from the anus. Anus with close-set anal fin base. Caudal fin forked, upper part slightly longer than the lower part. Upper and lower edges of the caudal peduncle with caudal adipose keels, upper adipose keel height mostly 1/2 of the caudal peduncle depth. Caudal peduncle length 90.2-119.0% of its depth. Body trunk covered by tiny scales, except for the ventral surface before the pelvic fin origin. Lateral line incomplete. Cephalic lateral line system with 3 + 3 supratemporal, 6 supraorbital, 3 + 8 infraorbital, and 7-11 preoperculo-mandibular pores.
Stomach U-shaped, intestine long, after stomach, with a bend. Swim bladder divided into two chambers. Anterior chamber covered by dumbbell-shaped bony capsule, and posterior chamber developed.
Coloration. Dorsal surface and trunk of body yellowish brown, abdomen gray and translucent, stomach and intestine visible from outside. Fin membrane hyaline.
Distribution and habitat. The new species was collected from Andong Township, Xincheng County, Laibin City, Guangxi Zhuang Autonomous Region, China (24 • 18.57 ′ N, 108 • 59.61 ′ E). Troglonectes canlinensis sp. nov. lives in a karst cave, where water accumulates to form a pool. Most specimens were collected in the rainy season. During the winter, the pool dries up and the cave opening is too narrow for human access. The water temperature was 20 • C during the survey period in July 2022.  Paratypes. KIZ-GXNU202207-09, 9 ex., 29.9-54.3 mm SL, collected with holotype. Diagnosis. Troglonectes canlinensis sp. nov., T. duanensis, T. lingyunensis, T. macrolepis, T. hechiensis, and T. retrodorsalis share their whole trunk being scaled, except for the head and area between the pectoral fins and pelvic fins; other species of Troglonectes have scaleless bodies or bodies scaled after the dorsal fin origin in Tr. furcocaudalis. However, the new species can be distinguished from T. duanensis by the incomplete lateral line (vs. absent), from T. lingyunensis and T. macrolepis by the eye being present (vs. eye reduced to black pigment), from T. hechiensis by the 8-10 inner-gill rakers on first gill arch (vs. 14), and from T. retrodorsalis by the tip of the anterior nostril being elongated to barbel-like and the nostril barbel length being nearly twice the nostril tube length (vs. nostril barbel length being less than 1/2 of the tube length).
Genetic comparisons. The molecular phylogenies based on BI analysis showed that Troglonectes species formed a monophyletic group, sister to the genus Paranemachilus. Mitochondrial differentiation. The pairwise comparisons of ctyb revealed that the average uncorrected p-distances interspecies of Troglonectes ranged from 0.2% to 12.2% (average 7.7%, Table 2). The maximum uncorrected p-distance was between Tr. jiarongensis and Tr. barbatus, and the minimum p-distance was between Tr. translucens and Tr. donglanensis. The average uncorrected p-distance between Tr. canlinensis sp. nov. and other congeneric species ranged from 3.0% to 9.0% (average 6.8%).

Discussion
The genus Troglonectes is currently distributed in the Pearl River system in Guangxi and Guizhou Provinces, and is endemic to China. Although Zhang et al. [2] mentioned that one of the identifying features of the genus is a forked caudal fin, there are truncated, emarginated, and forked caudal fins, three types of caudal fin. The phylogenetic tree indicates that the species of Troglonectes divided into sub-clade 1 contains species with truncated caudal fins and sub-clade 2 contains species with emarginated or forked caudal fins. Hence, the caudal fin shape and phylogenetic tree support that Troglonectes could be divided into two groups; the truncated caudal fin group contains Tr. hechiensis, Tr. retrodorsalis, and Tr. shuilongensis, and the emarginated or forked caudal fin group contains duanensis. Thus, based on our BI analysis and external characteristics, the genus description for Troglonectes includes the following characteristics: anterior and posterior nostrils separated by a short distance shorter than the diameter of the posterior nostril, tip of anterior nostril elongated to barbel-like, and adipose keels on the upper and lower edges of the caudal peduncle present.
Luo et al. [7] treated Tr. donglanensis and Tr. duanensis as synonyms of Tr. translucens, and Tr. jiarongensis and Tr. dongganensis as synonyms of Tr. elongatus based on morphological characteristics and a lack of genetic differences, respectively. Troglonectes dongganensis, Tr. elongatus, Tr. jiarongensis, and Tr. longibarbatus formed a monophyletic group in the phylogenetic tree, and the genetic distance was 0.  10−12); from Tr. hechiensis by the 8−10 inner-gill rakers on the first gill arch (vs. 14) and 9−10 branched pectoral fin rays (vs. 11); and from Tr. retrodorsalis by the 8−10 inner-gill rakers on the first gill arch (vs. 13−14) and 9−10 branched pectoral fin rays (vs. 11−12). Troglonectes canlinensis sp. nov. is different from Tr. translucens, Tr. jiarongensis, and Tr. lihuensis owing to its forked caudal fin (vs. emarginated) and scaled body (vs. scaleless); it can be further differentiated from Tr. jiarongensis and Tr. lihuensis by its incomplete lateral line (vs. absent in Tr. lihuensis and complete in Tr. jiarongensis) and upper adipose keel height being mostly 1/2 of the caudal peduncle depth (vs. equal to the caudal peduncle depth); and from Tr. jiarongensis by the 13−14 branched caudal fin rays (vs. 16). The new species is different from Tr. furcocaudalis owing to its whole body being scaled, except for the head and thorax (vs. scaled after the dorsal fin origin),  Species of Troglonectes are highly adapted to survive in cave habitats and are found only in limited regions with relatively small populations. Ma et al. [13] mentioned that cave fish have morphological adaptations to extreme cave environments, including the degeneration or disappearance of the eyes, reduced pigment, and scales. Additionally, cave fish have specialized features including well-developed tentacles and prolonged pectoral fins. Species of Troglonectes have developed barbels, well-developed adipose keel on the upper and lower caudal peduncles, reduced or no eyes, lateral line, scales, and pigment; these characteristics are adaptions to cave environments. As their life histories are limited to caves, these fish are vulnerable to various threats, such as habitat degradation, hydrological alterations, environmental pollution, resource overexploitation, and non-native species introduction [13]. Karst caves and subterranean streams are common geological features in Guangxi. More than 300 freshwater fish species have been recorded in Guangxi, including 61 cavefish [11]. On 16 September 2022, the Department of Forestry of the Guangxi Zhuang Autonomous Region published a list of wildlife under key protection in Guangxi, which included all cavefish species. The new species is currently only known from the type locality, where a few specimens were collected when the water rose from the cave during the rainy season. The discovery of this previously unknown species can hopefully lead to conservation measures to protect this area.

Conclusions
One new species of Troglonectes is described herein based on its morphological characteristics and molecular analysis. Additionally, the phylogenetic tree indicates species of Troglonectes divided into two sub-clades, viz. the truncated caudal fin sub-clade and emarginated or forked caudal fin sub-clade.

Nomenclatural Acts Registration
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