A new species of the genus Yunnanilus, designated Yunnanilus triangulus sp. nov., is described from the Nanpan River in Yunnan Province, southwestern China. Nuptial males of this species are distinguished by the presence of tubercles on the caudal peduncle during the reproductive period, a feature not observed in other members of the genus. The new species is further characterized by the following unique combination of morphological characters: body scaled, smaller eyes than interorbital width, nine inner gill rakers on first gill arch, lateral head length 27.1%–30.9% SL, head width 14.3%–17.2% SL, eye diameter 19.6%–23.8% of lateral head length, and caudal peduncle depth 94.0%–130.6% of its length. Morphological and molecular evidence support the validity of this species, despite its geographic proximity to Y. polylepis. These findings underscore the ecological specialization that exists within the genus Yunnanilus and highlight the urgent need for species-specific conservation strategies, given their restricted distributions and dependence on specific habitats.

Caudal peduncle with tubercles, freshwater fish, morphological characters, sexual dimorphism

Nichols (1925) designated Nemacheilus pleurotaenia Regan, 1904, as the type species of the nemacheilid loach subgenus Yunnanilus, with the following defining characters: short and compressed body, small scales, incomplete lateral line, slightly forked caudal fin, nostrils separated by greater distance than between posterior nostril and eye, and anterior nostril forming flap-like tube. Subsequently, Kottelat and Chu (1988) elevated Yunnanilus to the rank of genus, while Yang and Chen (1995) later categorized Yunnanilus species into two major assemblages, Y. nigromaculatus and Y. pleurotaenia species groups, based on the presence or absence of lateral and cephalic lateral line pores. Du et al. (2021) retained the Y. pleurotaenia group within Yunnanilus but reassigned the Y. nigromaculatus group to the revalidated genus Eonemachilus Berg, 1938. A more recent phylogenetic analysis of Chinese nemacheilids with tube-shaped anterior nostrils by Du et al. (2023) confirmed the generic status of Yunnanilus and refined its diagnostic characters to mouth inferior; anterior and posterior nostrils separated, anterior nostrils tube-like, without elongated barbel-like structure; cheeks scaleless; lips with furrows; and lateral line and cephalic lateral line pores present (Du et al. 2021, 2023).

Currently, Yunnanilus comprises 20 valid species, all restricted to lakes, marshes, and slow-flowing waters within Yunnan and Sichuan Provinces of southwestern China (Qin et al. 2024). Among these, six species are distributed in the Yangtze River system, including Y. discoloris Zhou & He, 1989, Y. longibulla Yang, 1990, Y. pleurotaenia (Regan, 1904), and Y. spanisbripes An, Liu & Li, 2009 in the Jinsha River; Y. sichuanensis Ding, 1995 in the Yalong River; and Y. jiuchiensis Du, Hou, Chen & Yang, 2018 in the Tuo River; and 13 species are distributed in the Pearl River basin, including Y. beipanjiangensis Li, Mao & Sun, 1994 in the Beipan River; Y. analis Yang, 1990; Y. chui Yang, 1991; Y. elakatis Cao & Zhu, 1989; Y. forkicaudalis Li, 1999; Y. macrogaster Kottelat & Chu, 1988; Y. macrolepis Li, Tao & Mao, 2000; Y. macrositanus Li, 1999; Y. nanpanjiangensis Li, Tao & Lu, 1994; Y. paludosus Kottelat & Chu, 1988; Y. parvus Kottelat & Chu, 1988; Y. polylepis Qin, Shao, Du & Wang, 2024; and Y. yangi He et al., 2024 in the Nanpan River, and one species, Y. chuanheensis Jiang, Zhao, Du & Wang, 2021 in the Red River (Lixian River) (Du et al. 2021; Qin et al. 2024).

In April 2018, eight specimens of Yunnanilus were collected from a tributary of the Nanpan River in Huaning County, Yuxi City, Yunnan Province, China. Morphological and molecular analyses confirmed that these specimens represent a previously undescribed member of the genus, which is herein described and compared with all known congeners.

All care and handling of experimental animals complied with the relevant laws of the Chinese Laboratory of Animal Welfare and Ethics (GB/T 35892-2018). Upon their collection, all specimens were rapidly euthanized by an overdose of anesthetic clove oil. The right-side pelvic fins of five individuals were removed and preserved in 99% ethanol for molecular analyses, and eight individuals were stored in 10% formalin for morphological comparisons. The specimens were deposited in the Kunming Natural History Museum of Zoology, Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS).

All counts and measurements followed the methodology described by Kottelat (1990). Initial data processing and preliminary statistical analyses were performed using Excel software. Genomic DNA was extracted from ethanol-preserved fin tissue, and partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) and cytochrome b (Cyt b) were sequenced by Tsingke Biotechnology Co., Ltd. (China). All sequences were assembled using SeqMan within the DNAStar package and aligned using MEGA v11.0 (Tamura et al. 2021). Sequences were submitted to GenBank (Accession Nos. PQ300642–PQ300646 for COI, PQ306053–PQ306057 for Cyt b). The phylogenetic position of Yunnanilus triangulus sp. nov. was determined using maximum-likelihood (ML) and Bayesian inference (BI) methods via the CIPRES Science Gateway (Miller et al. 2010). The ML tree was constructed using RAxML-HPC v8 (Stamatakis 2014) with a rapid bootstrapping configuration and 1000 bootstrap iterations. The BI tree was constructed using MrBayes in XSEDE v3.2.7a (Ronquist et al. 2012), with two simultaneous runs of 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 generate a consensus tree and estimate Bayesian posterior probabilities (BPPs). The resulting phylogenetic trees were viewed and edited using Figtree v1.4.4.

Molecular analysis unequivocally placed Yunnanilus triangulus sp. nov. within the genus Yunnanilus. This placement was further corroborated by several morphological features characteristic of the genus, including mouth inferior, lateral and cephalic lateral line pores present, anterior and posterior nostrils separated, an anterior nostril tube-shaped, and a tip not elongated into a barbel-like structure (Du et al. 2023; Qin et al. 2024).

The description of this new species increases the total number of Yunnanilus species native to the Yangtze, Pearl, and Red River basins to 21. Notably, Yunnanilus triangulus sp. nov. can be distinguished based on a combination of morphological characteristics related to the presence of scales, relative sizes of the eye diameter and interorbital width, absence of a processus dentiformis, lack of an outer gill raker on the first gill arch, and the specific number of branched caudal fin rays. The genus Yunnanilus can be broadly divided into two morphological groups based on the presence or absence of body scales. The ‘scaleless’ group contains Y. analis, Y. beipanjiangensis, Y. chui, Y. discoloris, Y. forkicaudalis, Y. paludosus, and Y. yangi, while the ‘scaled’ group includes the new species together with Y. chuanheensis, Y. elakatis, Y. jiuchiensis, Y. longibulla, Y. macrogaster, Y. macrolepis, Y. macrositanus, Y. nanpanjiangensis, Y. parvus, Y. pleurotaenia, Y. polylepis, Y. sichuanensis, and Y. spanisbripes. However, Yunnanilus triangulus sp. nov. can be distinguished from Y. elakatis, Y. macrositanus, Y. nanpanjiangensis, and Y. sichuanensis by whole body covered by scales, except head and thorax (vs. scales present only on caudal peduncle), from Y. longibulla and Y. jiuchiensis by eye diameter shorter than interorbital width (vs. longer), from Y. parvus, Y. macrogaster, Y. pleurotaenia, Y. macrolepis, Y. spanisbripes, and Y. longibulla by processus dentiformis absent (vs. present), from Y. macrolepis and Y. spanisbripes by outer gill raker on first gill arch absent (vs. present), and from Y. polylepis by body depth 20.0%–23.0% SL (vs. 15.0%–20.0%) and caudal peduncle depth 94.0%–131.0% of its length (vs. 73.0%–89.0%).

Sexual dimorphism is a common characteristic in members of the family Nemacheilidae. For example, males of the genus Oreonectes develop genital papillae posterior to the anus (Du et al. 2008); those of the genera Triplophysa and Barbatula exhibit thickened and widened outer branched pectoral-fin rays, accompanied by a slender pad of tubercles located anteroventral to the orbit (Zhu 1989). In Yunnanilus, sexual dimorphism is often expressed through color pattern variations. Although Qin et al. (2024) documented the presence of small tubercles on the body in nuptial males of Y. polylepis, with the presence of triangular tubercles on the caudal peduncle in nuptial males not previously reported. Zhang and Shen (1999) suggested that nuptial tubercles typically occur in areas of the body in contact during courtship or male-male competition, potentially serving to facilitate close physical interaction. Hence, we hypothesize that the caudal peduncle tubercles observed in Yunnanilus triangulus sp. nov. play a role in stimulating ovulation in females. However, further studies are necessary to validate this proposition.

Species of Yunnanilus are typically confined to small water bodies rich in submerged macrophytes, environments highly vulnerable to anthropogenic impacts and pollution. In September 2024, a survey of the type locality of Yunnanilus triangulus sp. nov. revealed that the species had disappeared in the pond, likely due to the rapid decline of submerged macrophytes and the introduction of large numbers of carp into the habitat. The sensitivity of Yunnanilus to water pollution, invasive species, and predation has led to consistent population declines across its range. These findings underscore the urgent need for targeted conservation strategies to safeguard Yunnanilus species and other freshwater fish in this ecologically significant karst region. It is hoped that this report will stimulate further research and conservation initiatives to address the growing threats faced by freshwater ecosystems in southwestern China.

M.L. and L.-N.D. measured the specimens, analyzed the data, conceived and designed the study, and prepared the manuscript, J.-B.L. analyzed the molecular data and constructed the phylogenetic tree. X.-Y.C. provided conceptualization and funding acquisition for the field survey and resources. All authors read and approved the final version of the manuscript.

This study was funded by the Project of the Innovation Team of Survey and Assessment of the Pearl River Fishery Resources (2023TD-10), Guangxi Natural Science Foundation Project (2022GXNSFAA035563), Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, China (ERESEP2022Z05), and the Position of Bioclassonomist of Chinese Academy of Sciences (CAS-TAX-24-054). We are grateful to L.L. Xu and H.D. Lyu for collecting specimens. We thank C. Watts for English corrections and suggestions. We thank C.Y. Lei for providing topotype specimens of Yunnanilus yangi. We especially thank help and supports from Bureau of Parks and Woods of Yuxi City throughout the field survey.