A new species of the genus Protocobitis is described based on morphological comparisons and molecular analyses from specimens of a subterranean tributary of the Hongshui River, Lingyun County, Baise City, and a cave in Jinya Township, Fengshan County, Hechi City, Guangxi, China. Both morphological and molecular data support the validity of Protocobitis longibarba sp. nov. The new species can be distinguished from congeners by the following combination of characteristics: whole body except for head and area between pectoral-fin and pelvic-fin origin sparsely covered with minute scales; barbels elongate; five or six branched pectoral-fin rays and four branched pelvic-fin rays; vertebrae 4+42. Maximum-likelihood and Bayesian-inference phylogenetic trees exhibited congruent topological structures, exhibiting high node support for the monophyly of Protocobitis longibarba (BPP = 1; BS = 100), which was clustered with the other congeners.

Cavefish, mitochondrial gene, Pearl River, taxonomy

The unique karst landforms of the Guangxi Zhuang Autonomous Region (hereinafter referred to as Guangxi) have resulted in extensive surface water and ground water systems, providing ideal conditions for the evolution and adaptative radiation of cavefish species. The perpetual absence of light in caves prevents photosynthesis, leading to a limited food supply primarily sourced from surface water exchange. This scarcity of food presents considerable challenges in providing adequate nutrition for fish reproduction. Consequently, populations of karst cavefish, such as Sinocyclocheilus hyalinus Chen & Yang, 1993, are extremely rare (Chen et al. 1994; Jeffery 2001, 2009; Zhao and Zhang 2009; Liang et al. 2011; Fan et al. 2024).

The genus Protocobitis Yang, Chen & Lan, 1994 was initially described based on specimens collected from Du’an County, Guangxi, with the type species Protocobitis typhlops Yang, Chen & Lan, 1994 (Yang et al. 1994). Endemic to Guangxi, this genus is a typical cave-dwelling fish species, displaying distinctive characteristics such as the absence of eyes, pigment degeneration leading to transparency or semitransparency, elongate barbels, reduction or absence of body scales, and tiny cranial bones (Zhao and Zhang 2006); thus, it demonstrates a high degree of adaptation to cave life. Four valid species have been recognized within the genus, including P. anteroventris Lan, 2013 and P. longicostatus Zhou, Qin, Du &Wu, 2024 from Baise City, P. typhlops from Hechi City, and P. polylepis Zhu, Lv, Yang & Zhang, 2008 from Nanning City (Yang et al. 1994; Zhu et al. 2008; Lan et al. 2013; Zhou et al. 2024). All known Protocobitis species are eyeless and exhibit varying degrees of rib degeneration.

Five specimens of Protocobitis were collected in February 2024 from a subterranean tributary of the Hongshui River in Luolou Town, Lingyun County, Baise City, and two collected in May 2024 from a cave in Jinya Township, Fengshan County, Hechi City, Guangxi, China. Results of our morphological and molecular analyses indicate that these loach specimens represent a new species of Protocobitis, which is described herein.

All field collections abided by the rules of the Fisheries Law of the People’s Republic of China. All activities conformed to the Laboratory Animal Guidelines for the Ethical Review of Animal Welfare (GB/T 35892-2018). After euthanizing the collected fish specimens with excessive anesthetic clove oil, the right pelvic fins were excised and placed in 95% alcohol for subsequent DNA sequencing, then the whole-body specimens fixed in 10% formalin and transferred to 75% ethanol for morphological study. Specimens were preserved at the Kunming Natural History Museum of Zoology, Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS), and Zhejiang Forest Resource Monitorign Center (ZJFRF), Hangzhou, Zhejiang. Counts and measurements followed Yang et al. (1994). All measurements were taken point-to-point with dial calipers to the nearest 0.1 mm. X-ray scanning and three-dimensional (3D) reconstructions were conducted using nano-computerized tomography (CT) with a GE V|tome|X m dual tube 300/180 kV system at the Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), CAS. The specimens were scanned with an energy beam of 80 kV and a flux of 80 × µA using 360° rotation, then reconstructed into a 4 096 × 4 096 matrix of 1 536 slices. The final CT reconstructed skull images were exported with a minimum resolution of 8.9 μm. Skull images were exported from the virtual 3D model reconstruction using Volume Graphics Studio v. 3.4.0.

The extraction, amplification, and sequencing of genomic DNA were conducted by Tsingke Biotechnology Co., Ltd (China). Partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) and cytochrome b (cyt b) were sequenced and submitted to GenBank (accession: PP866712–PP866715 for COI, and PP868402–PP868405 for cyt b). The sequencing results were manually checked, corrected, and assembled using SeqMan within the Lasergene v. 7.1.0 package, DNASTAR, Inc., Madison Wis. The assembled sequences were aligned using MEGA v. 7.0 (Kumar et al. 2016) for multiple comparisons, and redundant segments were trimmed to obtain consistent sequences for further analysis. Genetic diversity analyses and haplotype filtering were performed using DnaSP v. 5 (Librado and Rozas 2009).

The complete mitochondrial genomes of 14 cobitid species and two botiid species (Parabotia fasciata Dabry de Thiersant, 1872 and Leptobotia elongata Bleeker, 1870) were retrieved from GenBank to serve as the outgroup. The phylogenetic placement of Protocobitis longibarba was determined using maximum likelihood (ML) and Bayesian inference (BI) implemented in the CIPRES Science Gateway (Miller et al. 2010). The ML tree was reconstructed using RAxML-HPC v. 8 (Stamatakis 2014), with 1,000 rapid bootstrapping iterations. The BI tree was constructed using MrBayes in XSEDE v. 3.2.7a (Ronquist et al. 2012). Two parallel runs were performed, 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 sampled trees were discarded as burn-in, and the remaining trees were used to obtain a consensus tree and estimate Bayesian posterior probabilities (BPPs). The phylogenetic trees were viewed and edited using FigTree v. 1.4.4 (Rambaut 2009). Uncorrected pairwise distances (1000 replicates) based on concatenated dataset of mitochondrial COI and cyt b sequences was estimated using MEGA v. 7.0 (Kumar et al. 2016).

Both morphological and molecular data support the validity of Protocobitis longibarba. The genus Protocobitis is typically characterized by the presence of degenerate ribs, with P. anteroventris, P. longicostatus, and P. longibarba showing progressively shorter ribs and P. typhlops and P. polylepis lacking ribs entirely. Vertebral counts also show variation among the species, with P. anteroventris having the highest count (4+57), P. polylepis having the lowest count (4+38), and the other three species ranging from 4+42 to 4+43. The differences in vertebral count and rib degeneration indicated adaptations to cave environments. Morphologically, the new species can be distinguished from all other congeners based on a combination of the following characteristics: whole body covered with scales except for head and area between pectoral-fin origin and pelvic-fin origin (vs scaleless in P. anteroventris, scales present along midline of body in P. typhlops, 5–6 branched pectoral-fin rays (vs seven in P. anteroventris, P. longicostatus, and P. polylepis), and four branched pelvic-fin rays (vs five in other species within the genus). Furthermore, the new species can be distinguished from P. polylepis by the absence of pigmentation (vs black pigmentation present), from P. anteroventris and P. polylepis by caudal peduncle length 17%–20% of SL (vs 25%–28% in P. anteroventris and 15%–16% in P. polylepis), from P. longicostatus and P. typhlops by head height 50%–81% of lateral head length (vs 46%–50% in P. longicostatus and 52%–61% in P. typhlops), and from P. polylepis and P. typhlops by body height 8%–9% of SL (vs 17%–18% in P. polylepis and 9%–14% in P. typhlops).

This study provides a comprehensive morphological characters and x-ray scanning and three-dimensional (3D) reconstructions analysis of P. longibarba, contributing to the understanding of systematics and adaptations within this genus. The species exhibits distinct morphological characteristics, including rib reduction, which appears to be a consistent and diagnostic feature in Protocobitis. This structural adaptation may reflect an evolutionary shift away from reliance on ribs, potentially influencing body stability or flexibility. Additionally, we observed thickening of the chest and abdominal walls and absence of an air bladder, both of which are typical adaptations associated with benthic or bottom-dwelling species. The lack of an air bladder might indicate an ecological specialization, as reduced buoyancy is often advantageous for organisms that inhabit substrates or demonstrate sediment-burrowing behaviors (Longley 1993; Myhre and Klepaker 2009). These morphological traits raise the hypothesis that P. longibarba may engage in substrate penetration or mud-burrowing activities, behaviors warranting further ecological investigation to confirm.

Cavefish species exhibit high diversity, making them valuable for studying animal adaptations to extremely dark environments. Cavefish populations are extremely rare and highly sensitive to human disturbances due to their specialized habitats. Minor environmental changes, such as water pollution or extensive human activity, can lead to population extirpation or species extinction. During our field investigation, we observed that the karst cave inhabited by P. longibarba functions as a ponor cave in Lingyun City, leading to significant amounts of waste being transported into the cave by the river. This contamination has substantially impacted the habitat, hindering efforts to protect the cave-dwelling organisms. Consequently, establishing effective protection measures is crucial not only for preserving biodiversity but also for safeguarding the natural heritage and potential scientific value represented by cave-dwelling organisms.

We thank C. Watts for English corrections and suggestions. We thank S.P. Zhou for assistance in collecting specimens. We thank Y.M. Hou and P.F. Yi for their assistance with the CT scanning of the specimens.