Zoosyst. Evol. 100 (2) 2024, 747-754 | DOI 10.3897/zse.100.122962 > PENSUFT. nt BERLIN A new species of Yunnanilus (Cypriniformes, Nemacheilidae) from Yunnan, southwest China Zhi-Xian Qin!", Wei-han Shao*", Li-Na Du!*, Zhen-Xing Wang* 1 Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, Guilin, Guangxi 541004, China 2 Guangxi Key Laboratory of Rare and Endangered Animal Ecology, College of Life Science, Guangxi Normal University, Guilin, Guangxi 541004, China 3 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China 4 Guangxi Lujin Ecological Technology Company, Nanning, Guangxi, 530001, China https://zoobank. org/9ACE21C 4-8 D00-4C34-B7F5-EASFSFE07243 Corresponding authors: Li-Na Du (dulina@mailbox.gxnu.edu.cn); Zhen- Xing Wang (shangzhuxing@163.com) Academic editor: Nicolas Hubert ¢ Received 13 March 2024 Accepted 3 May 2024 Published 11 June 2024 Abstract A new species of Yunnanilus is described from the Nanpanjiang River, Yunnan, China. The new species, Yunnanilus polylepis, can be distinguished from other species of Yunnanilus by the following combination of characteristics: Processus dentiformis absent; eye diameter smaller than interorbital width; outer gill raker absent and 10 inner gill rakers on first gill arch; whole trunk covered by scales; nine branched dorsal-fin rays; 10 or 11 branched pectoral-fin rays; six branched pelvic-fin rays. Despite our phylogenetic analysis, which sheds light on the complex relationships among Yunnanilus species, the majority of Yunnanilus species are restricted to more localized environments and habitats. It is urgent to address the environmental threats that jeopardize their survival, especial- ly given their generally restricted distribution. Key Words Loach, mitochondrial gene, morphology, Nanpanjiang River, taxonomy Introduction Species belonging to the genus Yunnanilus Nichols, 1925 are primarily found in lakes, marshes, and slow-flowing waters, exhibiting an affinity for karstic regions, particu- larly in the Yunnan and Sichuan provinces of China (Du et al. 2021). Kottelat and Chu (1988), who identified eight valid species and six previously undescribed spe- cies from the Yunnan Plateau. Subsequently, Yang and Chen (1995) divided the species of Yunnanilus into the Y. nigromaculatus and Y. pleurotaenia species groups based on the absence or presence of lateral line and ce- phalic lateral line canals, respectively. Prokofiev (2010) * These authors contributed equally to this work. classified the family Nemacheilidae into five tribes, 1.e., Lefuini, Nemacheilini, Triplophysini, Vaillantellini, and Yunnanilini. However, the monophyly of the Yunnani- lini tribe was not supported in subsequent studies, with Du et al. (2021) and Luo et al. (2023) revising the classi- fication of Yunnanilini using both morphological charac- teristics and molecular evidence, resulting in the place- ment of the Y. nigromaculatus group into Eonemachilus Berg, 1938, ¥. pulcherrimus Yang, Chen & Lan, 2004 into Micronemacheilus Rendahl, 1944, Y. retrodorsalis (Lan, Yang & Chen, 1995) into Troglonectes Zhang & Zhao, 2016, and Y. jinxiensis Zhu, Du & Chen, 2009 into Paranemachilus Zhu, 1983. In addition, Du et al. Copyright Qin, Z.-X. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 748 (2023) delineated the phylogenetic relationships among Chinese nemacheilids possessing tube-shaped anterior nostrils, categorizing the spatial relationship between the anterior and posterior nostrils into three distinct types, 1.e., separated, adjacent, and closely set. Within this framework, Du et al. (2023) described a new genus, Guinemachilus Du et al., 2023, into which Y. bailianen- sis Yang, 2013 and Y. longibarbatus Gan, Chen & Yang, 2007 were placed. Subsequently, He et al. (2024) de- scribed a new species, Yunnanilus Yangi He et al., 2024, from Nanpan Jiang based on morphological and molec- ular data. Currently, 19 species of Yunnanilus have been recognized, with diagnostic characters including inferior mouth, anterior and posterior nostrils separated, anteri- or nostril base tube-shaped and tip without elongated barbel-like structure, and lateral line and cephalic later- al-line canals present (Kottelat and Chu 1988; Du et al. 2021, 2023). In November 2023, 13 Yunnanilus specimens were col- lected from a tributary of the Nanpanjiang River in Hua- ning County, Yuxi City, Yunnan Province, China. Based on morphological characteristics and molecular evidence, these specimens represent a previously undescribed spe- cies of Yunnanilus. Herein, we provide a description of the new species and its comparison to congeners. 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 were rapidly euthanized by an overdose of anesthetic clove oil soon after being collected. Five specimens were pre- served in 99% ethanol for molecular analyses, and eight specimens were stored in 10% formalin for morpholog- ical study. 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 Kottelat (1990). The data were initially processed using Excel software for preliminary statistical analysis. Genomic DNA was extracted from fins fixed in ethanol. Partial sequences of the mitochondrial cytochrome c oxidase subunit I (COT) and cytochrome b (cyt 6) were sequenced by Tsingke Biotechnology Co., Ltd. (China). All sequences were assembled by Seqman in the DNAstar software pack- age and aligned in MEGA v11.0 (Tamura et al. 2021). Sequences have been submitted to GenBank (Accession Nos. PP254216—254236 for COI, PP262976—62996 for cyt b). The phylogenetic position of Yunnanilus polyl- epis sp. nov. was determined by maximum likelihood (ML) and Bayesian inference (BI) methods, which were implemented in the CIPRES Science Gateway (Miller et al. 2010). The ML was constructed in RAXML-HPC v8 (Stamatakis 2014). Selected was the rapid bootstrap- ping configuration, and the bootstrapping iterations were 1000. The BI tree was conducted by MrBayes in XSEDE zse.pensoft.net Qin, Z.-X. et al.: A new species of Yunnanilus v3.2.7a (Ronquist et al. 2012). Two runs were performed simultaneously with four Markov chains starting from a random tree. The chains were run for five million gener- ations and sampled every 100 generations. The first 25% of sampled trees were discarded as burn-in, and the re- maining trees were used to create a consensus tree and estimate Bayesian posterior probabilities (BPPs). The constructed phylogenetic trees were viewed and edited by FigTree v1.4.4 (Rambaut 2009). Results Yunnanilus polylepis sp. nov. https://zoobank.org/B34187E3-BE59-4EC6-90A E-72F3266CFD89 Type materials. Holotype. K1Z2023000009 (Kunming Natural History Museum of Zoology, KIZ, CAS), female, 43.7 mm standard length (SL), Qixitan Park, Panxi Town, Huaning County, Yuxi City, Yunnan, P. R China; Nanpan- Jiang River; 24.2434°N, 103.1221°E, C.S. Yang collected in November 2023. Paratypes. Seven specimens. KIZ2023000010-14, female, 31.9-37.1 mm SL, KIZ2023000039-40, male, 30.3-31.8 mm SL; same as holotype. Other materials. DLN202301 80-184, preserved in 99% ethanol for molecular study, same as type specimens. Etymology. The specific name polylepis is derived from the characteristic of being entirely covered by scales Gender: Masculine. We suggest the Chinese and English common names as “% fis zs Fa fi’ and “densely scaled Yunnan loach,” respectively. Diagnosis. The new species is distinguished from all other members of the genus based on the following characters: whole trunk covered by scales; processus dentiformis absent; eye diameter smaller than interorbital width; nine branched dorsal-fin rays; 10 or 11 branched pectoral-fin rays; six branched pelvic-fin rays; outer gill raker absent and 10 inner gill rakers on first gill arch. Description. Morphometric and meristic data are giv- en in Table 1. Whole trunk covered with small and dense tubercles. Greatest body depth anterior to dorsal-fin or- igin, posterior portion gradually compressed from dor- sal-fin to caudal-fin base. Head length longer than depth and deeper than width. Snout slightly blunt, shorter than postorbital length of head. Eye diameter smaller than in- terorbital width, posterior nostril closer to anterior margin of eye than to tip of snout; anterior and posterior nostrils separated, distance greater than diameter of posterior nos- tril, base of anterior nostril tube-shaped, not elongated to barbel-like (Fig. 11). Body densely scaled except for head and thorax; pec- toral-fin origin to pelvic-fin origin covered by smaller and sparse scales. Upper jaw processus dentiformis absent. Three pairs of barbels, two rostral pairs and one maxillary pair; inner rostral barbel reaching posterior nostril; outer rostral barbel reaching anterior margin of eye; maxillary barbel reaching posterior margin of eye. Zoosyst. Evol. 100 (2) 2024, 747-754 Table 1. Morphometric and meristic data of Yunnanilus polyl- epis Sp. Nov. Characters Holotype Paratypes (Mean+SD) Total length (mm) 53.8 38.1-45.6 (42.2+2.8) Standard length (mm) 43.7 30.3-37.1 (33.9+2.6) Percent of standard length (%) Deepest body depth 15.7 16.3-19.6 (17.6+1.1) Head width 13.4 12.6-15.7 (13.9+1.1) Lateral head length 257. 25.2-28.2 (26.5+1.0) Prepelvic length 54.3 52.5-55.9 (54.3+1.4) Preanal length 75.4 72.9-78.4 (76.0+2.1) Preanus length 71.9 70.1-77.3 (73.7+2.3) Caudal-peduncle length 12.5 11.6-13.7 (13.0+0.8) Caudal-peduncle depth 10.1 10.0-11.5 (10.6+0.6) Percent of lateral head length (%) Head width 53.0 49.9-57.7 (52.4+2.9) Head depth 57.4 51.5-62.4 (57.9+4.1) Eye diameter 19.6 16.7-27.0 (21.8+3.3) Interorbital width 27.0 23.9-32.9 (29.4+3.0) Snout length 39.4 31.1-41.7 (37.7+4.2) Percent of caudal-peduncle length (%) Caudal-peduncle depth 80.8 73.2-88.7 (81.8+5.6) Dorsalfin rays iv, 9 iv, 9 Pectoral-fin rays Tog Pal i, 10-11 Pelvic-fin rays i, 6 i, 6 Anal-fin rays iii, 5 ill, 5 Branched caudaLfin rays 16 15 10 mm 749 Dorsal fin with four unbranched and nine branched rays; origin nearer to snout tip than to base of caudal fin; pectoral fin with one unbranched and 10 or 11 branched rays (mostly 10), inserted immediately anterior to vertical through posteriormost point of operculum; pelvic fin with one unbranded and six branched rays, tips of pelvic fin not reaching anus; anal fin with three unbranched and five branched rays, origin closer to anus; caudal fin emargin- ate, with 15 or 16 branched rays (mostly 15). Ten inner gill rakers, without outer gill rakers on the first gill arch; later- al line incomplete, with 15—20 lateral line pores, reaching between tip of pectoral-fin and dorsal-fin origin; cephalic lateral system with 13—14+3 infraorbital canal pores, 7-9 supraorbital canal pores, 6—8 supratemporal canal pores, and 9-10 preoperculomandibular canal pores. Stomach U-shaped, intestine long and_ straight (Fig. 2B). Swim bladder divided into two chambers, ante- rior chamber covered by dumbbell-shaped bony capsule, posterior chamber developed, connected with anterior chamber by slender tube, tube length about half of poste- rior chamber length (Fig. 2A). Coloration. In life, both sexes, head and trunk with gray- ish background color. Lower margin of eye to dorsal head surface dark brown, dorsal head with heart-shaped dark brown pattern, ventral head surface without color pattern. Figure 1. Morphometric characters of Yunnanilus polylepis sp. nov. A—C. Lateral, dorsal, and ventral views of female, holotype K1IZ2023000009; D—F. Lateral, dorsal, and ventral views of male, paratype KIZ2023000041; G—H. Living photo of female and male; I. Location of anterior and posterior nostrils. zse.pensoft.net 750 Predorsal trunk with five or six dark brown blotches, larger width than interspace. Four or five dark brown blotches after dorsal fin. Two elliptical, dark brown spots at base of dorsal fin, two dark brown spots at base of caudal fin. Fin rays with dark pigments, fin membrane hyaline. In females, upper line of flank with 12—14 dark brown large spots (Fig. 1G). In males, body with black longitudinal stripe on both sides (Fig. 1H). In formalin-fixed specimens, lateral stripe and blotches somewhat faded, body generally light yellow. Distribution and habitat. Yunnanilus polylepis sp. nov. is currently only known from Qixitan Park, Panxi Town, Huaning County, Yuxi City, Yunnan, China; Nan- panjiang River (24.2434°N, 103.1221°E). This species inhabits a deep pool with water depths ranging from 3 to 8 m, characterized by a rich presence of macrophytes (Fig. 3). Other fish species present in the pool include Discogobio brachyphysallidos and Sinocyclocheilus sp. Despite its confined distribution, the population of Yun- nanilus polylepis sp. nov. remains stable, largely due to the enforcement of a fishing ban within the park. anterior chamber stomach _>— intestine oy posterior chamber 5mm Qin, Z.-X. et al.: A new species of Yunnanilus Genetic comparisons. Of the 1737 bp in combined alignment, Y polylepis sp. nov. and Y. pleurotaenia were amplified in this study. These sequences were used for mo- lecular phylogenetic analysis together with 34 complete mitochondrial genomes and six cyt 6 sequences from Gen- Bank. Parabotia fasciata Dabry de Thiersant, 1872 and Leptobotia elongata (Bleeker, 1870), two botiid species, were used as outgroups. Given that BI and ML analyses produced overall identical topologies, only the BI tree with Bayesian posterior probabilities (BPP) and bootstrap sup- port (BS) values are presented here (Fig. 4). The phyloge- netic tree strongly supports samples of Yunnanilus polylepis sp. nov. to group into Yunnanilus. Furthermore, Yunnani- lus polylepis sp. nov. was identified as a sister to the clade containing Y analis, Y. chuanheensis, Y. jiuchiensis, and Y. pleurotaenia (BPP = 1; BS = 100). However, the molec- ular phylogenies do not support the monophyly of Yunnani- lus. Yunnanilus yangi was weakly supported to be a sister group to Eonemachilus (BPP = 59; BS = 61), and then clad- ed together with those specimens of Yunnanilus (Fig. 4). tubercles Figure 2. The air baldde (A), stomach and intestine (B), KIZ 2023000010, and tubercles on the trunk (C), KIZ2023000011 of Yun- nanilus polylepis sp. nov. A 98°E =: 99°E 100°E 9 101°E 102°E 103°E 104°E 105°E 106°E 98°E 99°E 100°E 9 101°E 102°E 103°E 104°E 105°E 106°E 1) \ Figure 3. Type locality of Yunnanilus polylepis sp. nov. A. Distribution map; B. Habitat photo of the type locality at the time of collection. zse.pensoft.net 751 Zoosyst. 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