Traccatichthys punctulatus is here described from the Liu-Jiang flowing into the Xi-Jiang of the Pearl River basin at Jinxiu County, Guangxi Zhuang Autonomous Region, southern China. It is distinguished from all congeners by having a unique body coloration of a series of brown spots along the lateral line and dorsal-fin. Other diagnostic characters, not unique to this new species, are caudal-fin and lateral body colorations, and absence of pectoral-fin tuberculation. Its validity is further confirmed by its monophyly recovered in a molecular phylogenetic analysis based on mitochondrial COI and Cyt b gene sequences and species-level divergence between the new species and its closest relatives.

Genetic divergence, morphological differentiation, species description, taxonomy

The loach genus Traccatichthys was originally established by Freyhof and Serov (2001) to include two species: a Vietnamese species, Nemacheilus taeniatus Pellegrin & Chevey, 1936 (type species), and a Chinese species (Micronemacheilus pulcher Nichols & Pope, 1927). Subsequent taxonomic revisions showed that this morphology-based Chinese species actually comprises multiple distinct species. Prokofiev (2004) described specimens from Sanya, Hainan Island, as a species (M. zispi) distinct from M. pulcher. While both species were referred to Traccatichthys, a third Chinese species of the genus (T. tuberculum) was described from a coastal river (Jian-Jiang) of Guangdong Province, southern China (Du et al. 2012). Outside China, M. bacmeensis (= T. bacmeensis) was described from northern Vietnam within the Red River basin by Hao and Binh in Nguyen (2005). Although Kottelat (2012, 2013) treated both T. zispi and T. bacmeensis as synonyms of T. taeniatus, Du et al. (2012) recognized T. zispi as valid while retaining T. bacmeensis as a synonym of T. taeniatus. Endruweit’s (2014) examination of the topotypical specimens of T. bacmeensis confirmed that this species is valid. Currently, the genus is composed of five valid species distributed across Guangdong, Guangxi Zhuang Autonomous Region (hereinafter referred to as Guangxi), Guizhou, Fujian, and Hainan Island in China, Vietnam, and Laos (Jin 1984; Kottelat 2001, 2013). Given that Traccatichthys species are a small-sized, with conserved morphology and intraspecific phenotypic variation, leading to considerable taxonomic confusion and disputed classification, the current taxonomy requires molecular scrutiny.

During our field surveys in 2005 and 2024, several specimens of Traccatichthys were collected from Jinxiu Yao Autonomous County, Guangxi Zhuang Autonomous Region, China. The collected specimens exhibited distinct morphological features that differed from previously described species within Traccatichthys. A combination of detailed morphological examinations and molecular phylogenetic analyses confirmed these specimens as a new species, which is described herein.

A small portion of the right pelvic fin was clipped for molecular analysis, with the remaining tissue preserved in vials containing 99.9% ethanol. Tissue samples from three individuals were used for mitochondrial cytochrome c oxidase subunit I (COI) and cytochrome b (Cyt b) gene sequencing. COI was amplified using the primers FISH F1 (5’-TCAACCAACCACAAAGACATTGGCAC-3’) and FISH R1 (5’-TAGACTTCTGGGTGGCCAAAGAATCA-3’), and the Cyt b gene was amplified using universal primers L14724 (5’-GACTTGAAAAACCACCGTTG-3’) and H15915 (5’-CTCCGATCTCC GGATTACAAGAC-3’). DNA was extracted using a Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech (Shanghai) Co., Ltd., China) following the manufacturer’s protocols. The polymerase chain reaction (PCR) was carried out in a total volume of 25 µL, including 12.5 µL of 2 × Taq mix, 1 µL of each forward and reverse primer, 1 µL of DNA template, and 9.5 µL of double distilled water (ddH₂O). Sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. (China).

The sequencing results were manually checked, corrected, and assembled using SeqMan in the Lasergene v7.1.0 package (DNASTAR, Inc., Madison, WI, USA). The assembled sequences were aligned using the MUSCLE algorithm in MEGA v11 (Edgar 2004; Tamura et al. 2021), with redundant segments trimmed to obtain consistent sequences and aligned sequences concatenated to form a combined COI and Cyt b data matrix, totaling 1,712 base pairs. The genome sequencing data were submitted to GenBank (Accession: PQ814574–PQ814590 for COI, PQ818764–PQ818780 for Cyt b).

The complete mitochondrial genomes of 17 Nemacheilidae species and two botiid species were retrieved from GenBank to serve as outgroup taxa. The phylogenetic placement of Traccatichthys punctulatus sp. nov. was determined using maximum-likelihood (ML) and Bayesian inference (BI) analyses, implemented using the CIPRES Science Gateway (Miller et al. 2010). The ML tree was constructed using RAxML-HPC v8 (Stamatakis 2014), with 1,000 rapid bootstrapping iterations. The BI tree was constructed using MrBayes in XSEDE v3.2.7a (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, with the first 25% of sampled trees discarded as part of the burn-in phase. The remaining trees were used to create a consensus tree and estimate Bayesian posterior probabilities (BPPs). The phylogenetic trees were visualized and edited using FigTree v1.4.4 (Rambaut 2009). Uncorrected pairwise distances (1000 replicates) based on mitochondrial COI sequences were estimated using MEGA v11 (Tamura et al. 2021).

All counts and measurements followed Kottelat (1990). Specimens examined were stored in the Guangxi Normal University (GXNU), Guilin; the Kunming Natural History Museum of Zoology at the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences, Kunming; and the Museum of Aquatic Organisms at the Institute of Hydrobiology (IHB), Chinese Academy of Sciences, Wuhan.

The new species forms a distinct clade in the phylogenetic tree, with genetic distances from other congeners ranging between 8.52% and 10.79%. The clear genetic and morphological separation of T. punctulatus sp. nov. from its congeners strongly supports its recognition as a distinct species. Genetic analyses validated the observed morphological differences, particularly the unique spotted patterning on the dorsal and caudal fins as well as the lateral body. Morphologically, this new species shares typical characteristics of Traccatichthys, including a complete lateral line, closely positioned anterior and posterior nostrils, papillated lips, and well-developed dentiform projections. However, the new species also possesses several unique traits that distinguish it from its congeners. Juveniles of T. punctulatus sp. nov. exhibit short, slightly separated lateral stripes that resemble those of T. zispi. By contrast, these lateral stripes in adults merge into serrated black blotches, with larger, irregularly sized spots along the lateral line and more prominent markings on the caudal peduncle. T. punctulatus sp. nov. exhibits irregular spots along the lateral midline extending from the operculum to the caudal-fin base, a character absent in other congeneric species. Additionally, the black blotch at the caudal-fin base is near-triangular, in contrast to the almost circular or rectangular spot seen in other species. These unique characteristics differentiate T. punctulatus sp. nov. from other species in the genus Traccatichthys.

In the dorsal fin, the unbranched rays are devoid of black pigmentation in the new species, distinguishing them from T. bacmeensis and T. pulcher, which possess such pigmentation. Instead of the marginal stripes observed in T. bacmeensis, T. pulcher, and T. tuberculum, the dorsal fin of T. punctulatus sp. nov. displays a row of irregular black spots. The anal fin exhibits a marginal black stripe, a characteristic absent in T. bacmeensis and T. tuberculum. In contrast to T. pulcher and T. tuberculum, the new species lacks marginal stripes along the upper and lower edges of the caudal fin. Instead, the caudal fin is covered with numerous irregular black spots. In T. taeniatus and T. zispi, however, all fins are unmarked and devoid of stripes. These pigmentation patterns collectively distinguish T. punctulatus sp. nov. from its congeners. Additionally, individual variation was observed on the dorsal surface anterior to the dorsal fin, with some specimens exhibiting stripes while others lacked any markings. The source of this variation remains unclear and may be influenced by environmental factors, genetic diversity, or developmental stages, warranting further investigation.

In the phylogenetic analysis, T. pulcher and T. taeniatus exhibit a close genetic relationship with a relatively low genetic distance (1.58%); however, they show significant differences in external morphology. T. taeniatus is distinguished from T. pulcher by the absence of distinct black submarginal streaks on the dorsal and caudal-fin lobes, which are present in T. pulcher. Additionally, T. pulcher has larger oval blotches on the caudal peduncle and along the lower edges of the body from the ventral to the caudal, whereas such markings are absent in T. taeniatus. In terms of geographic distribution, T. taeniatus is primarily found in Vietnam and Laos, with its type locality in Phú Thọ Province, northeastern Vietnam. In contrast, T. pulcher has a broader distribution, with its type locality in Hainan, China, and occurs mainly in Guangxi, Guangdong, and Hainan, as well as in coastal cities of Vietnam from the Chinese border to central Vietnam. This clear geographic separation, combined with morphological differences, supports their status as distinct species despite their relatively low genetic divergence. However, further studies integrating additional molecular markers, population-level sampling, and ecological data are needed to fully resolve their taxonomic relationship.

Until now, the genus Traccatichthys comprises six recognized species, including the newly described species. Unfortunately, due to the lack of molecular materials, T. bacmeensis was excluded from our phylogenetic analysis, and the phylogenetic status of this species still remains unknown. No specimens of this species are available in this study. Morphological comparisons of the new species are made with T. bacmeensis in terms of Endruweit’s (2014) redescription based on topotypic specimens.

There is controversy over the type locality of T. pulcher (Du et al. 2012). In terms of their original accounts, Nada Town and Sanya, Hainan Island, are the type localities for T. pulcher and T. zispi, respectively. However, the specimens we collected from Nada Town do not conform to T. pulcher (Figs 5, 6), but have no marginal and submarginal bands on dorsal fin and upper and lower marginal bands on the caudal fin, thus conspecific with T. zispi (Fig. 7). In contrast, specimens we collected from Dongfang City and Baisha Li Autonomous County exhibit the characters typical for T. pulcher. Thus, we agree with Du et al. (2012)’s third explanation that the type locality of T. pulcher requires further investigation.

Figure 5. 

Illustration of T. pulcher from the original description (Nichols and Pope 1927).

Figure 6. 

Lateral (a), dorsal (b), and ventral (c) views of T. pulcher from Baisha Li Autonomous County, Hainan Island, China. Scale bar: 10 mm.

Figure 7. 

Lateral (a), dorsal (b), and ventral (c) views of T. zispi from Nada Town, Hainan Island, China. Scale bar: 10 mm.

The electronic version of this article in portable document format represents a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new name contained in the electronic version is effectively published under the Code from the electronic edition alone (see Articles 8.5–8.6 of the Code). This published work and the nomenclatural acts it contains have been registered in ZooBank LSIDs (Life Science Identifiers) and can be resolved, and the associated information can be viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/.

Z.-X.Q. measured the specimens, analyzed the data, and prepared the manuscript. F.L. and L.-N.D. conceived and designed the study and reviewed and edited the manuscript. J.-J.Z. participated in the observation of the new species specimens. All authors read and approved the final version of the manuscript.

This study was funded by the Project of Financial Funds of the Ministry of Agriculture and Rural Affairs: Investigation of Fishery Resources and Habitat in the Pearl River Basin (ZJZX-10; ZJZX-04), the 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 National Natural Science Foundation of China (No. 31872202). We thank X. Wen, H.Y. Cheng, and L. Wei for their assistance in the measurement of the specimens. We thank D. M. Guo for providing a live photograph of this species. We are grateful to E. Zhang for the review of this paper.