A new species of Yunnanilus (Cypriniformes, Nemacheilidae) from Yunnan, southwest China

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, especially given their generally restricted distribution.


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, particularly 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 species 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 cephalic lateral line canals, respectively.Prokofiev (2010) classified the family Nemacheilidae into five tribes, i.e., Lefuini, Nemacheilini, Triplophysini, Vaillantellini, and Yunnanilini.However, the monophyly of the Yunnanilini tribe was not supported in subsequent studies, with Du et al. (2021) and Luo et al. (2023) revising the classification of Yunnanilini using both morphological characteristics and molecular evidence, resulting in the placement of the Y. nigromaculatus group into Eonemachilus Berg, 1938, Y. pulcherrimus Yang, Chen & Lan, 2004into Micronemacheilus Rendahl, 1944, Y. retrodorsalis (Lan, Yang & Chen, 1995) into Troglonectes Zhang & Zhao, 2016, and Y. jinxiensis Zhu, Du & Chen, 2009into Paranemachilus Zhu, 1983. In addition, Du et al. (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, i.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. bailianensis Yang, 2013and Y. longibarbatus Gan, Chen & Yang, 2007were placed. Subsequently, He et al. (2024) described a new species, Yunnanilus Yangi He et al., 2024, from Nanpan Jiang based on morphological and molecular data.Currently, 19 species of Yunnanilus have been recognized, with diagnostic characters including inferior mouth, anterior and posterior nostrils separated, anterior nostril base tube-shaped and tip without elongated barbel-like structure, and lateral line and cephalic lateral-line canals present (Kottelat and Chu 1988;Du et al. 2021Du et al. , 2023)).
In November 2023, 13 Yunnanilus specimens were collected from a tributary of the Nanpanjiang River in Huaning County, Yuxi City, Yunnan Province, China.Based on morphological characteristics and molecular evidence, these specimens represent a previously undescribed species 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 preserved in 99% ethanol for molecular analyses, and eight specimens were stored in 10% formalin for morphological 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 (COI) and cytochrome b (cyt b) were sequenced by Tsingke Biotechnology Co., Ltd.(China).All sequences were assembled by Seqman in the DNAstar software package 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 polylepis 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 bootstrapping configuration, and the bootstrapping iterations were 1000.The BI tree was conducted by MrBayes in XSEDE 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 generations and sampled every 100 generations.The first 25% of sampled trees were discarded as burn-in, and the remaining 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).Paratypes.Seven specimens.KIZ2023000010-14, female, 31.9-37.1 mm SL, KIZ2023000039-40, male, 30.3-31.8 mm SL; same as holotype.
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 "多鳞云南鳅" 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 given in Table 1.Whole trunk covered with small and dense tubercles.Greatest body depth anterior to dorsal-fin origin, posterior portion gradually compressed from dorsal-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 interorbital 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 nostril, base of anterior nostril tube-shaped, not elongated to barbel-like (Fig. 1I).
Body densely scaled except for head and thorax; pectoral-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.
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 emarginate, with 15 or 16 branched rays (mostly 15).Ten inner gill rakers, without outer gill rakers on the first gill arch; lateral 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, anterior chamber covered by dumbbell-shaped bony capsule, posterior chamber developed, connected with anterior chamber by slender tube, tube length about half of posterior chamber length (Fig. 2A).
Coloration.In life, both sexes, head and trunk with grayish 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.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; Nanpanjiang 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 Yunnanilus polylepis sp.nov.remains stable, largely due to the enforcement of a fishing ban within the park.

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 molecular phylogenetic analysis together with 34 complete mitochondrial genomes and six cyt b 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 support (BS) values are presented here (Fig. 4).The phylogenetic tree strongly supports samples of Yunnanilus polylepis sp.nov. to group into Yunnanilus.Furthermore, Yunnanilus 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 molecular phylogenies do not support the monophyly of Yunnanilus.Yunnanilus yangi was weakly supported to be a sister group to Eonemachilus (BPP = 59; BS = 61), and then claded together with those specimens of Yunnanilus (Fig. 4).

Discussion
Yunnanilus polylepis sp.nov.possesses typical characteristics of the genus Yunnanilus, including an inferior mouth, anterior and posterior nostrils separated, a tubeshaped base of anterior nostrils that is not elongated into a barbel-like structure, and lateral line and cephalic lateral-line canals present (Du et al. 2021(Du et al. , 2023)).

Figure 1 .
Figure 1.Morphometric characters of Yunnanilus polylepis sp.nov.A-C.Lateral, dorsal, and ventral views of female, holotype KIZ2023000009; 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.

Figure 3 .
Figure 3. Type locality of Yunnanilus polylepis sp.nov. A. Distribution map; B. Habitat photo of the type locality at the time of collection.

Figure 4 .
Figure 4. Bayesian phylogram of Yunnanilus based on a concatenated dataset of mitochondrial cytochrome c oxidase subunit I (COI) and cytochrome b (cyt b) sequences.The numbers on the branches represent BPPs from BI and bootstrap supports from ML.