Zoosyst. Evol. 100 (3) 2024, 941-963 | DOI 10.3897/zse.100.124370 ave Te BERLIN Taxonomic resolution of the hillstream suck-loach Beaufortia pingi species group (Cypriniformes, Gastromyzontidae) and two new species from Southwest China—Beaufortia granulopinna and Beaufortia viridis Jing-Chen Chen! *, Jia-Jia Lit'*, Wen-Qiao Tang!*, Xin-Rui Pu*, Hao-Tian Lei* 1 Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, Shanghai Ocean University, Shanghai 201306, China 2 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China 3 International College, Yunnan Agricultural University, Kunming 650500, China 4 College of Plant protection, China Agricultural University, Beijing 100093, China https://zoobank. org/5 9836C 29-33 F6-40F 1-AYEC-16D17086D820 Corresponding author: Wen-Qiao Tang (wqtang@shou.edu.cn) Academic editor: Nicolas Hubert # Received | April 2024 # Accepted 31 May 2024 Published 9 July 2024 Abstract Two new species, Beaufortia granulopinna and Beaufortia viridis, are described from the upper Pearl River system in southwest China. Both species share the characteristics of the Beaufortia pingi species group, including prominent vertical stripes on the lateral body and pinnate-type lower lips, distinguishing them from other Beaufortia species. Beaufortia granulopinna is differentiated from other species in the group by possessing a unique set of characteristics: the presence of well-developed prominent tubercles on the first 6—9 pectoral fin rays in adults; and a significant proportion (54.76%) of individuals experiencing blurriness or absence of ver- tical stripes in the mid-section of the lateral body upon reaching adulthood. The minimum interspecific genetic distance within the genus based on mitochondrial cytb gene sequences is 10.80%. Beaufortia viridis is distinguished from other species in the group by consistently exhibiting vertical stripes of uniform length, width, and spacing across all stages of growth; the absence of tubercles on the branched rays of pectoral fins; and a body coloration of dark cyan to green. The minimum interspecific genetic distance within the genus based on mitochondrial cytb gene sequences is 4.60%. Molecular phylogenetic results confirm that the Beaufortia pingi Species group forms a monophyletic clade, which is congruent with morphological classification findings. This study also addresses and resolves the taxonomic ambiguity surrounding Beaufortia pingi and Beaufortia zebroida, providing a redescription of these taxa. Key Words Beaufortia pingi, Beaufortia zebroida, Beaufortia, morphology, molecular phylogeny, redescription Introduction The genus Beaufortia, belonging to the order Cyprini- formes and family Gastromyzontidae, is distributed in the upper and middle reaches of the Yangtze River sys- tem, the Pearl River system, Hainan Island, and from Yunnan in China to the northern part of the Red River system in Vietnam (Kottelat 2012). This genus comprises small, benthic fish species that inhabit mountain streams. To adapt to fast-flowing environments, these rheophilic fish have undergone significant morphological special- izations, including a compressed body, a flattened ventral surface, and greatly expanded paired fins forming a suc- tion cup-like structure. These adaptations enable them to adhere to the rocky substrate, resisting the currents while feeding on algae and invertebrates. Copyright Chen, J.-C. 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. 942 In 1932, Hora established the genus Beaufortia for Gastromyzon species distributed in China, based on the absence of a continuous, elongated skin flap between the bases of the pectoral and ventral fins. Other characteris- tics of this genus as defined by Hora include small gill openings restricted to above the pectoral fins, the posteri- or edges of the ventral fins fused into a fin disk, a mouth slit smaller than 1/3 of the head width, and pectoral fins extending beyond the origin of the ventral fins (Hora 1932). These diagnostic features have been widely ac- cepted over time, and several species have subsequently been discovered and classified within this genus. At the onset of this study, the Catalog of Fishes had recorded 13 valid species within the genus, 12 of which were distributed in China (Frice et al. 2024). Among these, Beaufortia yunnanensis (Li, Lu & Mao, 1998) is particu- larly controversial. It was initially classified under the ge- nus Paraprotomyzon, and its type specimen has been lost; it was later reassigned to the genus Beaufortia by Kottelat (Li et al. 1998; Kottelat 2012). B. buas (Mai, 1978) was deemed questionable due to unclear locality data, low identification clarity in hand-drawn illustrations, and rudi- mentary morphological information (Kottelat 2012). His- torically, six other species have been included in the genus Beaufortia based on Vietnamese literature. Among these, B. fasciolata Nguyen, 2005; B. multiocellata Nguyen, 2005; and B. triocellata Nguyen, 2005 are now considered junior synonyms of B. zebroida (Fang, 1930) (Kottelat 2012). Gastromyzon daon Mai, 1978; Gastromyzon elon- gata Mai, 1978; and Gastromyzon loos Mai, 1978, which were described in the same publication as Beaufortia buas, also suffer from unclear locality data, low identification clarity in hand-drawn illustrations, and rudimentary mor- phological information and are currently regarded as spe- cies inquirenda and assigned to the genus Pseudogastro- myzon (Kottelat 2012). Kottelat (2013) included them as species inquirenda within the genus Beaufortia. According to Chen et al. (2023), species of the genus Pseudogastro- myzon are restricted to southeastern mainland China and do not extend to Vietnam. Therefore, the validity and taxo- nomic status of these species remain contentious. Following extensive sampling of specimens from the genus Beaufortia and through morphological and phylo- genetic analyses, we identified two new species: Beaufor- tia granulopinna Chen & Tang, 2024, sp. nov. and Beau- fortia viridis Chen & Tang, 2024, sp. nov. Additionally, we addressed the taxonomic issues surrounding Beaufor- tia pingi and Beaufortia zebroida, providing redescrip- tions of these two species. Materials and methods Sample collection Handheld dip nets were utilized as the primary tool for sample collection. According to documented literature, the distribution range of the B. pingi species group in- zse.pensoft.net Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group cludes Lingyun County, Tian’e County, Tianlin County, and Longzhou County in Guangxi Province, and Guang- nan County in Yunnan Province, where specimen collec- tion was conducted (Fang 1930; Chen 1990; Chen and Tang 2000). Due to various natural and anthropogenic fac- tors leading to habitat alterations, no specimens were col- lected in Tian’e County and Longzhou County. Extensive exploration of other potential distribution areas was con- ducted, leading to the discovery and collection of speci- mens in the Youjiang District of Baise City, Debao Coun- ty, Xilin County, Napo County, and Wuming District of Nanning City in Guangxi Province, and Wenshan Zhuang and Miao Autonomous Prefecture in Yunnan Province. Additional collections of B. szechuanensis (Fang, 1930), B. liui Chang, 1944, B. niulanensis Chen, Huang & Yang, 2009, B. polylepis Chen, 1982, B. kweichowensis (Fang, 1931), and B. /everetti (Nichols & Pope, 1927) specimens were conducted from the middle and upper reaches of the Yangtze River in Sichuan Province, the Jinsha River basin and Nanpan River basin in Yunnan Province, the Li River basin in Guangxi Province, and the Duliu River basin in Guizhou Province, as well as from Hainan Prov- ince for comparative purposes. Following euthanasia, specimens were preserved in a 95% ethanol or 0.3% for- malin solution, with 3—5 individuals randomly selected from each batch for mitochondrial cytb gene sequencing. Despite repeated attempts, no specimens of B. yunnanen- sis, B. huangguoshuensis Zheng & Zhang, 1987, B. in- termedia Tang & Wang, 1997, and B. cyclica Chen, 1980 were obtained, and morphological data were cited from their original descriptions based on examination of their holotypes or paratypes. All fish collections followed the Law of the People’s Republic of China on the Protection of Wildlife, and we strictly adhered to all applicable inter- national, national, and institutional guidelines for the care and use of animals. For other questionable species with unobtainable spec- imens and disputed taxonomic status, their original mor- phological descriptions were referenced. Morphometric data measurement Countable characteristics were examined using a stereo- scopic microscope. The last branched anal-fin and dor- sal-fin rays located on the last complex pterygiophore were counted as a single ray (Fig. 1). A considerable per- centage of the population belonged to a phenotype char- acterized by attenuated and unbranched terminal fin rays in the paired fins. This specific anatomical feature was quantitatively assessed as equivalent to 0.5 of a standard branched fin ray. Following the photographic documen- tation of specimens in dorsal, lateral, and ventral views, measurements were conducted using TPSDIG2 software (Rohlf 2016), as depicted in Fig. 1. Statistical analyses of the measurable characteristics were conducted using SPSS v.26.0 (IBM Corp. 2020) to assess the significance of differences. Zoosyst. Evol. 100 (3) 2024, 941-963 943 “TU =e en ee — Figure 1. A. Morphometric measurement methodology for species within the genus Beaufortia: | - Standard length (SL); 2 - Eye diameter (ED); 3 - Gill slit length (GSL); 4 - Pectoral fin base length (PBL); 5 - Head length (HL); 6 - Body depth (BD); 7 - Dorsal fin base length (DBL); 8 - Anal fin base length (ABL); 9 - Post-anal fin length (PoAL); 10 - Caudal peduncle length (CL); 11 - Caudal peduncle depth (CD); 12 - Head length (HL); 13 - Snout Length (SnL); 14 — Head width (HW); 15 - Interorbital distance (JOD); 16 - Distance between nostrils (ND); 17 - Pre-dorsal Length (PrDL); 18 - Pectoral fin disc length (PL); 19 - Head width at pectoral fin origin (HWPO); 20 - Mouth slit width (MW); 21 - Pectoral fin disc width (PW); 22 - Body width (BW); 23 - Pelvic fin disc width (PvW); 24 — Pre-pectoral length (PrPL); 25 — Pre-pelvic length (PrPvL); 26 - Pelvic fin disc length (PvL); 27 - Pre-anal pore length (PrApL); 28 - Pre-anal fin length (PrAL); B. The last branched anal-fin and dorsal-fin rays located on the last complex pterygiophore were counted as a single ray. Mitochondrial cytb gene sequencing DNA was extracted from the right pectoral fin rays us- ing a kit produced by Sangon Biotech (Shanghai) Co., Ltd., following the protocol provided. Primers GAC TTG AAG AAC CAC CGT TGT TAT T 5’-3’ and TCT TCG GAT TAC AAG ACC GAT GCT TT 5’—3’ were employed to amplify the cytb gene. The PCR mixture consisted of luL DNA template, 1 uL of each primer (10 uM), 12.5uL Taq Mix (Sangon Biotech), and 9.5uL deionized water, totaling a reaction volume of 25uL. The PCR conditions included an initial denaturation at 95 °C for 3 minutes, followed by 35 cycles of 94 °C for 30 seconds, 55 °C for 45 seconds, and 72 °C for 60 seconds, with a final exten- sion at 72 °C for 5 minutes and a hold at 4 °C. Amplified products were sequenced by Sangon Biotech, and the sequences were verified for accuracy before submission to the National Center for Biotechnology Information (NCBI). The collection sites for the molecular materials are listed in Table 1. Phylogenetic analysis and genetic distance estimation A total of 35 mitochondrial cytb gene haplotypes were identified. The cytb sequence of Plesiomyzon baotin- gensis (KF732713), retrieved from NCBI, was utilized as the outgroup, with a haplotype of B. szechuanensis (NC027291) included as additional material. Phylogenetic analyses were conducted using PHYLOSUITE (Zhang et al. 2020) with sequence alignment performed by MAFFT (Katoh and Standley 2013) set to automatic strategy and normal alignment mode. ModelFinder (Kalyaanamoor- thy et al. 2017) was used to determine the best-fit mod- els for both Maximum Likelihood (ML) and Bayesian Inference (BI) methods. The ML phylogenetic tree was constructed using IQ-TREE (Nguyen et al. 2015), with the best-fit model according to BIC being MGK+G4, and the outgroup set to Plesiomyzon baotingensis, with de- fault parameters for other settings. The BI phylogenetic tree was constructed using MRBAYES 3.2.6 (Ronquist zse.pensoft.net 944 Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group Table 1. Collection localities and accession numbers of molecular samples. Species Specimen voucher Sampling localities River system GenBank accession Beaufortia granulopinna Sp. nov. SHOU20240103028 Tianlin County, Guangxi Pearl River (upper reaches) PP482589 SHOU20240102801 Tianlin County, Guangxi PP482590 SHOU20240103650 Tianlin County, Guangxi PP482592 SHOU20240103652 Tianlin County, Guangxi PP482593 $HOU20240115013 Guangnan County, Yunnan PP482591 Beaufortia viridis sp. nov. $HOU20240103615 Nanning City, Guangxi PP482580 SHOU20240126801 Lingyun County, Guangxi PP482581 SHOU20240112104 Guangnan County, Yunnan PP482578 SHOU20240111616 Guangnan County, Yunnan PP482579 SHOU20240112103 Guangnan County, Yunnan PP482582 Beaufortia pingi SHOU20240102207 Lingyun County, Guangxi PP482563 $HOU20240102216 Lingyun County, Guangxi PP482564 $HOU20240102217 Lingyun County, Guangxi PP482565 SHOU20240102206 Lingyun County, Guangxi PP482566 $HOU20240102602 Lingyun County, Guangxi PP482567 SHOU20240126802 Baise City, Guangxi PP482561 SHOU20240102212 Baise City, Guangxi PP482568 SHOU20240103210 Debao County, Guangxi PP482562 Beaufortia zebroida SHOU20240111304 Guangnan County, Yunnan Red River (lower reaches, North bank) PP482585 $HOU20240111302 Guangnan County, Yunnan PP482588 $HOU20240111313 Malipo County, Yunnan PP482586 SHOU20240111312 Funing County, Yunnan PP482587 Beaufortia kweichowensis $HOU20231212604 Congjiang County, Guangxi Pearl River (upper reaches) PP482574 $HOU20231212645 Lingchuan County, Guangxi PP482576 $HOU20231212620 Huizhou City, Guangdong Pearl River (lower reaches) PP482575 Beaufortia leveretti $HOU20231212641 Baisha County, Hainan Nandu River PP482577 Beaufortia szechuanensis SHOU20231220642 Neijiang City, Sichuan Yangtze River (upper reaches) PP482569 Beaufortia niulanensis SHOU20240120617 Qujing District, Yunnan PP482583 SHOU20240120616 Qujing District, Yunnan PP482584 Beaufortia polylepis SHOU20231220644 Kaiyuan City, Yunnan Pearl River (upper reaches) PP482570 SHOU20231220619 Shizong County, Yunnan PP482571 SHOU20231 220630 Luoping County, Yunnan PP482572 SHOU20231 220636 Luoping County, Yunnan PP482594 Beaufortia cf. szechuanensis SHOU20231220611 Qijiang District, Chongqing Yangtze River (upper reaches) PP482573 et al. 2012), with the model set to HKY+F+G4 and the Type material. Holotype. SHOU20240103001, outgroup as Plesiomyzon baotingensis, maintaining de- fault parameters for other settings. The resulting tree files were visualized and embellished using FIGTREE v1.4.4 (Rambaut 2018). Genetic distances were calculated using MEGAI11 (Tamura et al. 2021) with the Kimura 2-parameter model (rates among sites: G4), computing distances among all samples, average interspecific genetic distances, and av- erage intraspecific genetic distances. Results Taxonomic account Beaufortia granulopinna Chen & Tang, sp. nov. https://zoobank.org/8A80A35D-C2A 8-4B55-80D 1-2BFDBA68B374 Figs 2-4 Gastromyzon pingi Fang, 1930: 35-36, No. 955, paratype, former Lingyun County, near the border of Yunnan. Beaufortia pingi: Chen and Zhang (2006): 376-377, Tianlin County, Guangxi (fig. X98). zse.pensoft.net 72.48 mm total length (TL), 57.17 mm standard length (SL), adult (Fig. 2). Collected by Jing-Chen Chen and Qian- Yu Liang on 24 December 2023, from Lizhou Riv- er, a stream tributary of Bo'ai River of Pearl River basin, at Tianlin County, Guangxi Province, China (24°20.34'N, 106°21.624'E; c. 470m a.s.1.) (Fig. 5). Paratypes. 21 specimens from the same locality as ho- lotype, SHOU20240103002-022, 21.41-54.00 mm SL, were collected by Qian-Yu Liang and Jing-Chen Chen on 24 December 2023; 12 specimens from Guangnan County, Wenshan Zhuang, and Miao Autonomous Pre- fecture, Yunnan Province, China, SHOU20240115001- 012, 31.86-44.07 mm SL, were collected by Xinrui Pu and Jing-Chen Chen on 08 January 2024. Additional materials. Seven specimens from the type locality, SHOU20240103023-29, were collected by Qian- Yu Liang from December 2022 to April 2023; one specimen from Guangnan County, Wenshan Zhuang, and Miao Autonomous Prefecture, Yunnan Province, China, SHOU20240115013 was collected by Lin Yang and Lao Xing in December 2022. Diagnosis. B. granulopinna sp. nov. shares typical characteristics with members of the B. pingi species group, Zoosyst. Evol. 100 (3) 2024, 941-963 945 Figure 2. Lateral (top), dorsal (middle), and ventral (bottom) views of Beaufortia granulopinna sp. nov., holotype, adult, SHOU20240103001, 57.17 mm SL; from Lizhou River, a stream tributary of Bo'ai River of the Pearl River basin, at Tianlin County, Guangx! Province, China. Sub-adult Figure 3. Lateral (top), dorsal (middle), and ventral (bottom) views of Beaufortia granulopinna sp. nov. during early juvenile and sub-adult stages. A. Early juvenile stage, SHOU20240103658, from type locality, 16.09 mm SL, pelvic fins completely separated; B. Sub-adult stage, SHOU20240103005, paratype, 24.21 mm SL. zse.pensoft.net 946 with distinct vertical stripes on the flank, and a pinnate-type lower lip (vs. lacking prominent vertical stripes and having a dicot-type lower lip in other congeneric species apart from this group) (see Figs 6D, 7, Table 2). B. granulopin- na can be clearly distinguished from B. pingi, B. zebroida, and B. viridis sp. nov. by the presence of well-developed tubercles on the anterior 6—9 pectoral fin rays (vs. absent or inconspicuous tubercles on pectoral fin rays). Moreover, B. granulopinna can be distinguished from B. pingi by generally fewer branched fin rays in the paired fins, with 18.5—22 (mean 20.11+0.77) in pectoral fins (vs. 21-24 (mean 22.19+0.78)) and 15—18 (mean 16.51+0.86) in pel- vic fins (vs. 17-21 (mean 18.64+0.97)). It can be further differentiated from B. viridis sp. nov. and B. zebroida by a certain proportion (54.76%) of individuals exhibiting blurred or vanished vertical stripes in the mid-section of the lateral body in adulthood (vs. stable presence of verti- cal stripes at all growth stages). Description. Dorsal i1i-6—7(6.98+0.15), anal 11-4, pec- toral 1-18.5-22(20.11+0.77), pelvic 1-15-18(16.51+0.86). Lateral-line canal pores and scales: 63-73 (68.76+2.5) (see Table 2). Morphometric measurements for the specimens exam- ined are given in Table 3. See Fig. 2 for lateral, dorsal, and ventral views of the body. Head-thorax cylindrical, dorsal slightly humped me- dially, flattened ventrally, body compressed from pelvic fins to caudal peduncle. Head broad, blunt, length slight- ly less than width, more than depth, nuptial tubercles well-developed on lower half of head in mature individ- uals, snout rounded, length about half of head length. Mouth inferior, narrow, width about one-fifth head width, oN Tianlin County, Guangxi (Paratype) Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group horseshoe-shaped, angle about 96° from midpoint to ends. Upper lip smooth, without distinct papillae; lower lip pinnate-type, slightly concave medially with multi- lobed sides; jaw edges slightly protrude. Shallow groove between upper lip and snout, extending to mouth corners. Lateral grooves shallow or indistinct. Two pairs of rostral barbels present, with outer pair slightly longer, space be- tween rostral barbels with leaf-like folds, edges of which rounded and poor developed. One pair of maxillary bar- bels, length about equal to outer rostral barbels. Nostrils with tubular nasal flaps, distance between nostrils equals one-third head width. Eyes supralateral, medium-sized, eye diameter about one-quarter head length, interorbit- al space flat, width about half head width. Gill opening small, about equal to eye diameter, originated about verti- cally above the second branched pectoral fin ray, limited to dorsal side of head. Scales small, diameter smaller than pupil, dorsal surface of head, base of paired fins, and ven- tral area before pelvic fin bases nude. Lateral line com- plete, at midlateral. Dorsal fin base about equal to pre-pectoral length, starting around midpoint from snout to caudal fin base, adpressed extending to about midway between dor- sal fin origin and caudal fin base. Anal fin base length about half of that of dorsal fin base, adpressed extend- ing slightly beyond caudal fin base. Paired fins extending outwards, forming disc-like structure with body. Pecto- ral fin base slightly longer than head length, starting at the posterior one-third point of head, pectoral fin length about twice head length, tips of which reaching pelvic fin base midpoint, pectoral disc width about 1.5 times head width at pectoral origin. Anterior 6—9 pectoral fin rays Guangnan County, Yunnan (Paratype) Figure 4. Lateral (top), dorsal (middle), and ventral (bottom) views of one common phenotypic variation of Beaufortia granulopinna sp. nov. and other regional phenotypes. A. Phenotype with persistent vertical stripe on lateral body, paratype, SHOU20240103003, 40.77 mm SL, from Tianlin County, Guangxi; B. Regional phenotype from Qingshui River, Guangnan County, Yunnan Province, paratype, SHOU20240115001, 39.01 mm SL. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 941-963 Table 2. Key diagnostic features and countable characteristics of species within the genus Beaufortia. Species B. granulopinna Chen & Tang 2024, sp. nov. B. viridis Chen & Tang 2024, sp. nov. B. pingi (Fang, 1930) B. zebroida (Fang, 1930) ? B. fasciolata Nguyen, 2005 ? B. triocellata Nguyen, 2005 ? B. multiocellata Nguyen, 2005 B. szechuanensis (Fang, 1930) B. liui Chang, 1944 B. huangguoshuensis Zheng & Zhang, 1987 B. polylepis Chen, 1982 B. intermedia Tang & Wang, 1997 B. niulanensis Chen, Huang & Yang, 2009 ? B. yunnanensis (Li, Lu & Mao, 1998) B. leveretti (Nichols & Pope, 1927) B. kweichowensis (Fang, 1931) B. cyclica Chen, 1980 ? B. buas (Mai, 1978) ? B. daon (Mai, 1978) ? B. elongata (Mai, 1978) ? B. loos (Mai, 1978) Vertical Type of Pectoral branch Pelvic branch stripes on flank present present present present present present present absent absent absent absent absent absent absent absent absent absent absent absent absent absent lower lip pinnate pinnate pinnate pinnate pinnate pinnate pinnate Dicot Dicot Dicot Dicot Dicot Dicot Dicot Dicot Dicot Dicot rays Range Mean = 18.5- 22 19-23 21-24 20-23 21 21-22 21-22 Le 26.5 21 ay? 26-27 25-28 2030= a 26 23-29 24- 25.5 30 24 25 27 25 SD 20.11 + OFZ 21.09 + 103 22.19 + 0.78 21.5+ 0:73 21 24.4 + 0.89 24.9 + O59 30 24 25 27 25 Dorsal branch rays rays Range Mean Range Mean + SD + SD 15-18 16.51+ 6-7 698+ 0.86 0.15 14.5- 17.11 + Z: 7 19 1.02 17-21 1864+ 7-8 7.08+ 0.97 0.27 16-19 17.53 + 7 7 0.86 16 16 7 7 16-17 - y/ 7 15-16 - 7 7 18.5- 19.64 7 7 20.5 0.82 1¥. li 7 19 17 6 6 19-21 20+ 6-7 6.5 + 1.41 0.71 24 - 7-8 - 20 20 7 7 20 20 8 8 19.5- 21.54 8 8 23.5 1.77 21.5- 23+ 8 8 24.5 1.22 20 20 7 7 19 19 8 8 19 19 9 9 20 20 8 8 21 21 8 8 Anal branch rays Range Mean + SD 4 4 4 4 3-4 3.98+ 0.14 3-4 3.94+ 0.24 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4-5 44x 0.55 4-5 44+ 0.55 5 5 5 5 5 5 5 5 5 5 947 Lateral-line N canal-pores and scales Range Mean + SD 63-73 68.76+ 42 259 65-83 73.51+ 36 4.26 68-92 75.28+ 50 4.01 70-84 75.12+ 17 3195 87-88 — 2 86-96 - 3 91-102 - ri 82-95 894+ 5 Sey 92 92 1 84 84 1 80-84 82+ 2 2:83 81-86 - 9 78-90 84.33+ 3 6.03 87 87 il 59-63 60.6+ 5 1.82 60-64 624+ 5 ov 72 72 1 55-65 - - 75-85 - - 65 65 - Species belonging to the Beaufortia pingi species group are highlighted in bold; a question mark “?” indicates substantial taxonomic controversy surrounding the species, and “-” stands for no corresponding data. w= ex es Pap ‘a wu <2 'Nis Figure 5. Collection site of the holotype (SHOU20240103001) of Beaufortia granulopinna sp. nov., from Lizhou River, a stream tributary of Bo'ai River of the Pearl River basin, Tianlin County, Guangxi Province, China. Photographed by Qian-Yu Liang on 24 December 2023. zse.pensoft.net 948 Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group Table 3. Morphometric results for species within the Beaufortia pingi group. B. viridis sp. nov. (N=36) B. pingi (N=50) B. zebroida (N=17) All materials Range Mean + SD 18.82- 41.64+11.37 Range MeansSD Range MeanzSD 27.04- 47.5346.94 33.24- 41.2545.18 Characters B. granulopinna sp. nov. (N=42) Holotype All materials Holotype Range Means SD Standard length (mm) 57.17 21.41- 36.344+8.30 38.03 Sy albys Vertical stripe blurring 54.76% 0 propotion (N=23) Ratio of standard length to Body depth 3.97 3.97-6.27 5.10+0.43* 5.36 Body width 3.93 3.92-5.34 4.61+0.35 4.54 Head length 4.94 4.19-5.05 4.63+0.21 4.40 Caudal peduncle length 17.17 10.42- 14.38+2.61 SAL 21.40 Caudal peduncle depth 7.67 7.32-9.89 8.36+0.52* 9.51 Predorsal length 2.03 1.88-2.11 1.97+0.06 1.99 Prepelvic length 2.40 2.14-2.40 2.29+0.06 2.34 Prepectoral length 7.36 5.70-7.73 6.72+0.51 6.83 Pectoral base length 4.46 4.00-5.46 4.67+0.37 4.48 Pectoral length 2.56 2.44-3.56 2.63+0.19 2.47 Pectoral width 2.30 2.21-2.97 2.55+0.18 2.59 Pelvic length 3.27 = 3.03-3.88 3.46+0.20 3.30 Ratio of caudal peduncle length to Caudal peduncle depth 0.45 0.37-0.80 0.60+0.11 0.73 Ratio of head length to Head depth 1.05 1.01-1.66 1.27+0.12 1.54 Head width 0.71 0.71-0.91 0.82+0.05 0.87 Snout length 2.08 1.97-2.77 2.31+0.18 2.24 Eye diameter 5.15 3.37-5.23 4.30+0.50 441 Interorbital width 1.38 1.35-2.38 1.73+0.26 1.60 Prepectoral length 1.49 1.26-1.65 1.45+0.10 1.55 Ratio of head width to Mouth width 4.59 4.27-6.40 5.19+0.46 4.99 Internostril width 3.97 2.80-4.37 3.56+0.38 3.12 Ratio of postanal length to Caudal peduncle depth 1.21 1.06-1.49 1.29+0.11* 1.67 Ratio of prepelvic length to Length of pelvic origin to 1.01 0.97-1.21 1.07+0.06 1.08 anal origin 59.85 4.25-6.11 3.79-5.46 3.91-4.98 9.72- 17.16 7.89- 11.53 1.81-2.14 2.07-2.52 5.54-7.51 3.09-0/37 2.37-2.84 2.18-2.80 2.97-4.04 0.53-1.17 1.19-1.72 0.70-0.97 1.89-2.59 3.51-6.95 1.48-1.99 1.38-1.75 4.32-6.60 3.09-4.07 1.17-2.08 0.95-1.33 5.31+0.54 4.43+0.41 4,.45+0.24 12.5321.78 9.87+1.01 1.98+0.07 2.28+0.08 6.71+0.41 4.55+0.29 2.59+0.12 2.44+0.15 S88t012Z5 0.81+0.16 1.41+0.13 0.84+0.06 2.24+0.19 4.59+0.76 1.68+0.14 1.51+0.08 5.44+0.63 3.5140.27 1.61+0.22 58.12 94.00% (N=47) 3.91-6.48 3.65-4.88 4.42-5.32 10.85- L729 8.52- 193 1.95-2.21 2.19-2.49 6.60- 8.24* 4.06-5.24 2.43-2.84 2.13-3.11 2.94-3.67 0.57-0.99 1.05-1.58 0.65-0.88 1.76-3.24 3.86-5.58 1.18-2.30 1.39-1.68 4.69-6.72 2.69-4.53 1.29-1.86 5.09+0.71* 4.20+0.36* 4.9+0.18* 139721207 10.21+1.00 2.04+0.06 2.36+0.08 7.42+0.39* 4,.59+0.29 2.63+0.09 2.43+0.21 3.26+0.18 0.75+0.12 1.26+0.13 0.77+0.06 2.09+0.22 4.53+0.41 1.51+0.24 1.5140.07 5.79+0.50* So LHO:35 1.55+0.15 49.96 0 4.90-5.98 3.93-9, 19 4.28-5.06 9.62- 16.94 8.84-10.9 1.90-2.47 2,15-2:38 5.94-7.52 4.32-5.3 1.94-2.73 2.34-2.85 3.29-3.65 0.61-1.13 1.13-1.72 0.78-1.07 2.15-2.49 3.62-5.35 1.47-2.32 1.30-1.62 431-5,13 216-3:79 1.38-1.86 5.47+0.33 4.62+0.37 4.61+0.19 12372185 10.01+0.48 2.01+0.13 2.28+0.06 6.84+0.42 4.80+0.28 2.50+0.18 2.59+0.18 3.44+0.11 0.82+0.12 1.35+0.15 0.88+0.06 2.30+0.10 4.36+0.47 1.7840.28 1.49+0.09 5.02+0.36 Po yz 06 1.66+0.15 1.1140.10 0.90-1.27 1.00+0.07* 1.02-1.29 1.11+0.07 “*” denotes that, ina one-way ANOVA test, post-hoc comparisons reveal that the trait in question significantly differs from those of other species (without “*”’) (p < 0.05). with nuptial tubercles in adults. Pelvic fin shorter than pectoral, well-developed fleshy flap at dorsal base, last 1-3 branched rays partially connected by fin membrane forming pelvic disc, connected part about two-thirds ray length, remaining parts separated, forming notch in mid- dle rear edge, exposing anus. Pelvic disc width about equal to length. Anus at or near posterior edge of pelvic disc, distance to which less than to anal fin origin. Caudal fin length about equal to pelvic fin, slanted end, lower lobe slightly longer. Coloration in preservation. Preserved specimens from sub-adult to adult stage, body dark brown to grey, white ventrally. Head with black spots or vermiculations dorsally, 2—5 larger black blotches along mid-dorsal body anterior to dorsal fin. Sides with 4-15 thick dark vertical stripes, stripes posterior to dorsal fin origin wider than intervals. In smaller individuals, stripes clear and distin- guishable, in larger individuals, stripes anterior to caudal peduncle sometimes blur or disappear. Paired fin with white margin, black arc, or dotted-arc line inside white zse.pensoft.net ring. Dorsal fin with alternating black, white stripes, anal fin hyaline with black stripes. Coloration in live. In life, dorsal body dark brown to green. Mature individuals with metallic, green longitudi- nal band along lateral line, area below lateral line behind pelvic fins and base of paired fins sometimes light orange, more pronounced in Qing Shui River basin populations, Yunnan. Dorsal fin black pattern wider than white. Juvenile morphology. Pelvic fins completely separated, dorsal body side gray-brown, few wider vertical dark stripes on flank, 2—3 vertical black stripes on caudal fin, other fins hyaline or with inconspicuous black lines (see Fig. 3). Sexual dimorphism. In fully mature individuals, males slightly larger than females, with well-developed nuptial tubercles (see Fig. 8E, F). Geographic variation. Qing Shui River basin popula- tions, Yunnan, more pronounced orange color below lat- eral line behind pelvic fins, dorsal side of paired-fins base after sexual maturity compared to type locality Tianlin County populations (see Fig. 4B). Zoosyst. Evol. 100 (3) 2024, 941-963 949 lf Figure 6. Pinnate-type lower lip of the Beaufortia pingi species group, elongated, with the lower lip on both sides being multilobed, typically comprising more than three lobes, with deep divisions; the extremities of the lower lip extend to the corners of the mouth, with several wart-like protrusions present on the extended parts. The red line outlines the left side of the lower lip contour; the red arrow indicates the position of the ventral head sensory canal-pores located anterior to the origin of the pectoral fins. A. B. pingi, from the type locality; B. B. zebroida, from Napo County, Guangxi Province; C. B. viridis, from the type locality; D. B. granulopin- na, from the type locality. , Figure 7. Dicot-type lower lip of species outside the Beaufortia pingi group, shorter, with the sides of the lower lip being smooth or featuring 1—2 shallow notches; the ends of the lower lip do not extend to the corners of the mouth, or the extension is poor-developed and lacks protrusions. The red line outlines the left side of the lower lip contour; the red arrow indicates the position of the ventral head sensory canal-pores, with the second pair of canal-pores located posterior to the origin of the pectoral fins in all species except B. liui. A. B. leveretti, from the type locality; B. B. kweichowensis, from Congjiang County, Guizhou Province, downstream near the type locality; C. B. cyclica, holotype; D. B. intermedia, paratype; E. B. polylepis, syntype; F. B. huangguoshuensis, syntype; G. B. szechuanensis, from Neijiang City, Sichuan Province; H. B. niulanensis, from the type locality; I. B. /iui, syntype. (C-F and I were photographed by Yi-Yang Xu, Institute of Hydrobiology, Chinese Academy of Sciences.) zse.pensoft.net 950 Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group Figure 8. Sexual dimorphism within the Beaufortia pingi species group, with males exhibiting well-developed and numerous nup- tial tubercles on the head compared to females. A, B. B. viridis sp. nov.; C, D. B. pingi; and E, F. B. granulopinna sp. nov. Individual variation. Among the 42 specimens exam- ined, one (SHOU20240103017) with six branched rays in dorsal fin. Ethology. Inhabits shallow streams with rapid currents and smooth pebble substrates that adhere to crevices be- tween stones. Feeds on algae and small invertebrates and consumes mucus from fresh fish carcasses. Exhib- its strong territorial behavior and aggression; adults may head-butt and extend dorsal fins to drive away rivals. Distribution. Found exclusively in the small trib- utaries of the Bo'ai River basin, ranging from the zse.pensoft.net northwestern part of Guangxi to the eastern part of Yunnan in China, as well as in the small tributaries of the Qingshui River section and its downstream Nanpan River basin, upper reaches of the Pearl River system (secrrisg: 0): Etymology. The specific epithet granulopinna com- bines Latin “granulo-,’ meaning grainy, and “-pinna,” meaning fin, referring to the well-developed tubercles on pectoral fins; the term is in the nominative masculine singular. We propose the Chinese common specific name Se Zoosyst. Evol. 100 (3) 2024, 941-963 951 ; oak Ae ee OB. granulopinna sp. nov. : OB. viridis sp. nov. N OB. ping OB. zebroida = River ==== Border r 3500 = -200 50 Kilometers Figure 9. Distribution map of species within the Beaufortia pingi species group. Light red: B. granulopinna sp. nov.; light green: B. viridis sp. nov.; light blue: B. pingi; light yellow: B. zebroida. Rivers are labeled with their names in Chinese: Qingshui River ( JS 7KYL), Nanpan River (P43 #tYL), Beipan River (AE#tYL), Hongshui River (2E7K3*]), Tuoniang (GKURYL), Xiyang River (FUYFYL), Leli River (Ak {234]), Chengbi River (4 324"]), Bo’ai River (3!) bmyA]), Jian River (4371), You River (47), Wuming River (UMS JAY), Yu River (iS), Shuikou River (7k HyF], known as Song Bang in Vietnamese), and Red River (2135). Beaufortia viridis Chen & Tang, sp. nov. https://zoobank. org/268BD22A-C14B-4662-BFFC-A8C3369DACC7 Figs 10-12 Beaufortia pingi: Yue (1981): 170-172, Lingyun County, Guangx1, ad- mixed within B. pingi. Zheng (1989): 257-258, Lingyun County, Guangxi, admixed within B. pingi. Beaufortia pingi: Chen (1990): 85-87, Xiyang River, Guangnan Coun- ty, Yunnan. Beaufortia zebroidus: Chen and Tang (2000): 504-505, Tian'e County, Guangxi. Chen (2013): 292, Xiyang River, Yunnan. Type material. Holotype. SHOU20240103101, 48.42 mm total length (TL), 38.03 mm standard length (SL), adult (Fig. 10). Collected by Jing-Chen Chen and Qian-Yu Li- ang on 27 December 2023, from Wuming River, a stream tributary to the You River of the Pearl River basin, at Wuming District, Nanning City, Guangxi Province, China (24°20.34'N, 106°21.624'E; c. 160 m a.s.1.) (Fig. 13). Paratypes. 15 specimens from the same locality as holotype, SHOU20240103102-116, 18.82-46.64 mm SL, were collected by Qian-Yu Liang and Jing-Chen Chen on 27 December 2023; eight specimens from Lingyun County, Baise City, Guangxi Province, Chi- na, SHOU20240126101-108, 50-59.85mm SL, were collected by Qian-Yu Liang and Jing-Chen Chen on 24 January 2024; five specimens from Guangnan Coun- ty, Wenshan Zhuang, and Miao Autonomous Prefec- ture, Yunnan Province, China, SHOU20240109101- 105, 36.23—54.20mm SL, were collected by Lao Xing on 28 December 2023. Additional materials. Seven specimens from Guang- nan County, Wenshan Zhuang, and Miao Autonomous Pre- fecture, Yunnan Province, China, SHOU20240112101- 107, 39.97-48.64mm SL, were collected by Lin Yang and Lao Xing from December 2022 to February 2023. Diagnosis. B. viridis sp. nov. shares the typical charac- teristics with members of the B. pingi species group, with distinct vertical stripes on lateral body and a pinnate-type lower lip (vs. lacking prominent vertical stripes and hav- ing a dicot-type lower lip in other congeneric species apart from this group) (see Figs 6C, 7, Table 2). B. viridis sp. nov. can be clearly distinguished from B. granu- lopinna, B. pingi, and B. zebroida by consistent vertical stripes with uniform length, width, and inter-spacing at all growth stages (stripes on mid-section lateral body oc- casionally shorten in individuals from the Xiayang and Tuoniang Rivers in Yunnan) (vs. a certain proportion (54.76%) of adults exhibit blurred or vanished vertical stripes on mid-section lateral body in B. granulopinna, zse.pensoft.net 952 Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group Figure 10. Lateral (top), dorsal (middle), and ventral (bottom) views of Beaufortia viridis sp. nov., holotype, adult, SHOU20240103101, 38.03mm SL; from Wuming River, a stream tributary to the You River of the Pearl River basin, at Wuming District, Nanning City, Guangx! Province, China. stripe length and width uneven, with a high proportion (94.00%) of adults showing blurred or vanished mid-sec- tion vertical stripes in B.pingi; stripe length and width uneven, typically narrower than inter-spacing, sometimes reduced to dots in B. zebroida). B. viridis sp. nov. can be further distinguished from B. granulopinna sp. nov. by the lack of prominent tubercles on the branched rays of the pectoral fins (vs. well-developed tubercles on anterior 6-9 rays of the pectoral fins present). Moreover, B. viridis sp. nov. differs from B. zebroida by body dark cyan to green in dorsal profile (vs. brown yellow to golden). Description. Dorsal 111-7, anal 11-4, pectoral 1-19-23 (21.08+1.02), pelvic 1-14.5—19 (17.11+1.01). Lateral-line canal pores and scales: 65—83 (73.47+4.21) (see Table 2). Morphometric measurements for the specimens exam- ined are given in Table 3. See Fig. 10 for lateral, dorsal, and ventral views of the body. zse.pensoft.net Head-thorax cylindrical, dorsal slightly humped me- dially, flattened ventrally, body compressed from pelvic fins to caudal peduncle. Head broad, blunt, length slight- ly less than width, more than depth, nuptial tubercles well-developed on lower half of head in mature individ- uals, snout rounded, length about half of head length. Mouth inferior, narrow, width about one-fifth head width, horseshoe-shaped, angle about 94° from midpoint to ends. Upper lip smooth, without distinct papillae; lower lip pin- nate-type, slightly concave medially with multilobed sides; jaw edges slightly protrude. Shallow groove between up- per lip and snout, extending to mouth corners. Lateral grooves shallow or indistinct. Two pairs of rostral barbels present, with outer pair slightly longer, space between ros- tral barbels with leaf-like folds, edges of which rounded and poor developed. One pair of maxillary barbels, length about equal to outer rostral barbels. Nostrils with tubu- Zoosyst. Evol. 100 (3) 2024, 941-963 Early juvenile 953 Sub-adult Figure 11. Lateral (top), dorsal (middle), and ventral (bottom) views of Beaufortia viridis sp. nov. during early juvenile and sub- adult stages. A. Early juvenile stage, SHOU20240103623, from type locality, 12.64 mm SL, pelvic fins completely separated; B. Sub-adult stage, SHOU20240103102, paratype, 25.81 mm SL. lar nasal flaps, distance between nostrils equals one-third head width. Eyes supralateral, medium-sized, eye diameter about one-quarter head length, interorbital space flat, width about half head width. Gill opening small, about equal to eye diameter, originated about vertically above the second to third branched pectoral fin ray, limited to dorsal side of head. Scales small, diameter smaller than pupil, dorsal sur- face of head, base of paired fins, and ventral area before pelvic fin bases nude. Lateral line complete, at midlateral. Dorsal fin base about equal to pre-pectoral length, starting around midpoint from snout to caudal fin base, adpressed extending to about midway between dorsal fin origin and caudal fin base. Anal fin base length about half of that of dorsal fin base, adpressed extending to slightly beyond caudal fin base. Paired fins extending outwards, forming disc-like structure with body. Pectoral fin base slightly longer than head length, starting at the posterior one-third point of head, pectoral fin length about twice to head length, tip of which reaching pelvic fin base mid- point, pectoral disc width about 1.5 times head width at pectoral origin, tubercles on rays absent. Pelvic fin short- er than pectoral, with well-developed fleshy flap at dor- sal base, last 1-3 branched rays partially connected by fin membrane forming pelvic disc, connected part about two-thirds ray length, remaining parts separated, form- ing notch in middle rear edge, exposing anus. Pelvic disc width about equal to length. Anus at or near posterior edge of pelvic disc, distance to which less than to anal fin origin. Caudal fin length about equal to pelvic fin, slanted end, lower lobe slightly longer. Coloration in preservation. Preserved specimen from sub-adult to adult stage, body dark green to grey, white ventrally. Head with black spots or vermiculation dorsally, 2-5 larger black blotches along mid-dorsal body anterior to dorsal fin. Sides with 9-15 thick dark verti- cal stripes, with uniform length, width, and inter-spacing, easily recognizable at all ages. Paired fin with pale-white or hyaline margin, inner edges with continuous or dotted black arc. Dorsal fin hyaline with black stripes, one black spot present on the root before the second branched ray. Coloration in live. In life, sides of body dark cyan to green. After maturity, black stripes and spots on body turn metallic green, inter-spacing and hyaline fin membranes pale yellow. Juvenile morphology. Pelvic fins completely separat- ed, body dark green in dorsal profile, few wider vertical dark stripes on sides, 2—3 vertical black stripes on caudal fin, other fins hyaline or with inconspicuous black lines (see Fig. 11). Sexual dimorphism. [In fully mature individu- als, males generally slightly larger than females, with well-developed tubercles (see Fig. 8A, B). Geographic variation. From east to west, vertical stripes generally tend to become thinner. Specimens from type locality, inter-spacings narrower than dark stripes, sometimes appearing fissure-like (see Fig. 10). In contrast, Lingyun County populations typically ex- hibit broader and taller bodies, shorter and thicker caudal peduncles, longer snouts, and wider inter-spacings (see Fig. 12A, Table 4). In populations from the Xiyang River and Tuoniang River, vertical stripes vary greatly, some- times becoming finer, inter-spacings wider than stripes, few individuals with shorter stripes before caudal pedun- cle (see Fig. 12B). zse.pensoft.net 954 Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group Lingyun County, Guangxi (Paratype) Guangnan County, Yunnan (Paratype) Figure 12. Lateral (top), dorsal (middle), and ventral (bottom) views of different regional phenotypes of Beaufortia viridis sp. nov. A. Regional phenotype from Lingyun County, Guangxi, SHOU20240126104, paratype, 58.71 mm SL; B. Regional phenotype from Tuoniang River, Guangnan County, Yunnan, SHOU20240109105, paratype, 40.15 mm SL (left); SHOU20240109103, paratype, 39.36 mm SL (right). Table 4. Morphological variations among populations of Beaufortia viridis sp. nov. and Beaufortia pingi from different distribution areas. B. viridis sp. nov. Wuming County (N=16) Linyun County (N=8) Tuoniang River and Xiyang River (N=12) Characters Range Mean + SD Range Mean + SD Range Mean + SD Pectoral branched rays 19-21 20.25+0.68 21-22 21.37+0.44 21-23 22+0.74 Pelvic branched rays 14.5-17 16.5+0.71 17-19 17.62+0.74 16-19 17.58+1.10 Ratio of prepelvic length to Head width 1.53-1.88 1.65+0.08 1.40-1.65 1.53+0.08* 1.54-1.85 1.67+0.12 Pectoral width 1.01-1.30 1.11+0.07 0.94-1.03 0.99+0.03* 0.96-1.19 1.08+0.07 Body width 1.79-2.63 2.04+0.22 1.62-1.82 1.73+0.07* 1.79-2.29 1.97+0.15 Body depth 2.19-2.77 2.50+0.17 1.76-2.20 1.98+0.15* 2.09-2.65 2.34+0.21 Head depth 2.53-3.27 2.89+0.22 2.30-2.67 2.48+0.12* 2.40-3.14 2.79+0.25 Caudal peduncle depth 4.07-5.16 4,54+0.29 3.26-4.11 3.59+0.28* 3.90-5.05 4.55+0.37 Pelvic width 1.31-2.02 1.43+0.17 1.15-1.30 1.25+0.05* 1.21-1.74 1.44+0.17 Ratio of head length to Snout length 2.23-2.59 2.40+0.11* 1.89-2.12 2.01+0.08* 1.98-2.43 2.19+0.12* Ratio of postanal length to Caudal peduncle depth 1.48-2.08 1.10+0.16 1.17-1.39 1.28+0.08* 1.37-1.94 1.70+0.15 Ratio of prepelvic length to Length of pelvic origin to anal origin 1.06-1.33 1.19+0.74* 0.95-1.17 1.02+0.71 0.96-1.17 1.08+0.58 B. pingi Linyun County & Youjiang County (N=38) Debao County (N=12) Characters Range Mean + SD Range Mean + SD Pelvic branched rays 17.5-21 18.95+0.89 17-18.5 17.67+0.39 Ratio of pectoral width to Body depth 1.67-2.50 2.02+0.21 1.92-2.49 2.32+0.19** Ratio of postanal length to Caudal peduncle depth 1.29-1.77 1.50+0.13 1.58-1.86 1.69+0.09** Ratio of prepelvic length to Length of pelvic origin to anal origin 0.90-1.27 0.98+0.66** 0.97-1.14 1.07+0.52 “*” imdicates that, ina one-way ANOVA test, post-hoc comparisons show significant differences in the trait in question from populations in other regions (p < 0.05). “**? Indicates that, in an independent sample T-test, the trait in question significantly differs from populations in other regions (p < 0.01). Individual variation. Two specimens, SHOU20240109101 and SHOU20240112102, from Tu- oniang River, Guangnan County, Yunnan, vertical stripes notably shortened. One specimen, SHOU20240109701, from Chengbi River, Lingyun County, Guangxi, possess- es completely separated pelvic fins. Ethology. Inhabits shallow streams with rapid currents and smooth pebble substrates that adhere to crevices between stones. Feeds on algae and small invertebrates and consumes mucus from fresh fish carcasses. Exhibits territorial behavior. zse.pensoft.net Distribution. Found exclusively in small tributaries from the Tuoniang River, Xiyang River, Leli River, and Wuming River to the You River basin, the upper reaches of the Pearl River system, central Guangxi, and eastern Yunnan, China (see Fig. 9). Etymology. Species epithet “viridis,” from Latin, meaning “green,” describes bright green coloration over dorsal body after sexual maturity, in nominative mas- culine. We propose the Chinese common specific name “2x DME ea aK.” Zoosyst. Evol. 100 (3) 2024, 941-963 Za? ae i Ry oy 955 Figure 13. Collection site of the holotype (SHOU20240103101) of Beaufortia viridis sp. nov., from Wuming River, a stream trib- utary to the You River of the Pearl River basin, Wuming District, Nanning City, Guangxi Province, China. Photographed by Jing- Chen Chen on 27 December 2023. Beaufortia pingi (Fang 1930) Fig. 14 Gastromyzon pingi Fang, 1930: 31-34 (original description: former Lingyun County, near the border of Yunnan and Guangxi; Damaop- ing Village, Lingyun County, Guangxi). Nichols (1943): 231. Beaufortia pingi: Hora (1932): 319. Gastromyzon pingi: Nichols (1943): 231. Beaufortia pingi: Chen (1980): 113-114 (Xi River, Guangx1). Comparative materials. 23 specimens from type locality, SHOU20240102201-217, SHOU20240126201-206, 27.64— 56.89 mm SL, were collected by Qian-Yu Liang and Jing- Chen Chen from December 2022 to January 2024; 15 speci- mens from Youjiang District, Baise City, Guangxi Province, China, SHOU20240110201-204, SHOU20240127201-211, 43.53-56.28 mm SL, were collected by Qian-Yu Liang and Jing-Chen Chen from December 2021 to January 2024; 12 specimens from Debao County, Baise City, Guangxi Prov- ince, China, SHOU20240112201-202, SHOU20240103201- 210, 44.53-58.12 mm SL, were collected by Qian- Yu Liang from April 2023 to January 2024. Diagnosis. B. pingi shares the typical characteristics with members of the B. pingi species group, with dis- tinct vertical stripes on the flank and a pinnate-type low- er lip (vs. lacking prominent vertical stripes and having a dicot-type lower lip in other congeneric species apart from this group) (see Figs 6A, 7, Table 2). B. pingi can be clearly distinguished from B. granulopinna sp. nov. by absent or inconspicuous tubercles on pectoral fin rays (vs. well-developed tubercles present on anterior 6—9 rays of pectoral fins); paired fins with more branched rays, with 21—24 (mean 22.19+0.78) in pectoral fins and 17— 21 (mean 18.64+0.97) in pelvic fins (vs. 18.5—22 (mean 20.11+0.77) and 15-18 (mean 16.51+0.86)). B. pingi can be clearly distinguished from B. viridis sp. nov. by vertical stripes uneven in length and width and a signifi- cant proportion (94.00%) of adults displaying blurred or vanished stripes on the mid-section flank (vs. the stable presence of uniform vertical stripes in length, width, and inter-spacing at all growth stages). B. pingi differs from B. zebroida by having a larger proportion of adults with blurred or vanished mid-section vertical stripes (vs. sta- ble presence of vertical stripes) and body dark brown to green in dorsal profile (vs. brown yellow to golden). Redescription. Dorsal 111-7—8 (7.08+0.27), anal 11-3—4 (3.98+0.14); pectoral i-21—24 (22.19+0.78); pelvic 1-17- 21 (18.64+0.97). Lateral-line canal pores and scales: 68— 92 (75.28+4.01). Morphometric measurements for the specimens exam- ined are given in Table 3. See Fig. 14 for lateral, dorsal, and ventral views of the body. Body closely resembles B. viridis sp. nov. in general Shape and structure but significantly different in stripes pattern and coloration. Tubercles on rays absent or poor developed, only faintly visible in large males. Coloration in preservation. Preserved specimens from sub-adult to adult stage, body dark brown to grey, white ven- trally. Head with black spots or vermiculation dorsally, 2—5 larger black blotches along mid-dorsal body anterior to dor- sal fin. Sides of body with 4-15 vertical dark stripes, length and width of stripes uneven. In smaller individuals, stripes clear and distinguishable, in larger individuals, a significant proportion possesses a blur region before caudal peduncle, extending to dorsal fin origin at maximum, caudal peduncle with 2—5 distinct vertical stripes. Paired fin margins hyaline or pale white, inner edges with continuous or dotted black arc. Dorsal fin hyaline, with black stripes, one black spot on the root before the first branched ray. Coloration in live. In life, dorsal body dark brown to green. Mature individuals with metallic green longitudi- nal band along lateral line, area below lateral line behind zse.pensoft.net 956 Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group Debao County, Guangxi Lingyun County, Guangxi (Type locality) Figure 14. Lateral (top), dorsal (middle), and ventral (bottom) views of different regional phenotypes of Beaufortia pingi. A. Region- al phenotype from Lingyun County, Guangxi, SHOU20240126202, from type locality, 56.89mm SL. B. Regional phenotype from Debao County, Guangxi, SHOU20240103203, 58.12 mm SL (left), with clearer vertical stripes; SHOU20240103204, 53.18 mm SL (right), with blurred vertical stripes. pelvic fins and base of paired fins sometimes light orange, fin membranes pale green. Sexual dimorphism. In fully mature individuals, males slightly larger than females, with well-developed nuptial tubercles (see Fig. 8C, D). Geographic variation. Compared to type locality, Lingyun County, Guangxi, population in Debao County, Guangxi with more compressed body, and more elongat- ed tail (see Fig. 14B, Table 4). Individual variation. Among 50 _— specimens, four (SHOU20240102213, SHOU20240102215, SHOU20240126205, and SHOU20240127211) with eight dorsal fin-branched rays; one (SHOU20240127204) with three anal fin-branched rays. Ethology. Inhabits shallow streams with rapid currents and smooth pebble substrates that adhere to crevices be- tween stones. Feeds on algae and small invertebrates and consumes mucus from fresh fish carcasses. Exhibits terri- torial behavior and aggression; adults may head-butt and extend dorsal fins to drive away rivals. Distribution. Found exclusively in the small tributar- ies of the Chengbi River and Jian River to the You River basin, upper reaches of the Pearl River system, Western Guangxi, China (see Fig. 9). Beaufortia zebroida (Fang 1930) Fig. 15 Gastromyzon pingi zebroidus Fang, 1930: 35 (original description: Donggui River, Longzhou County, Guangx!). Beaufortia zebroidus: Hora (1932): 319. Gastromyzon pingi zebroidus. Nichols (1943): 231. Beaufortia pingi: Nguyen and Nguyen (2005): 282—283 Beaufortia fasciolata Nguyen & Nguyen, 2005: 588-590. Beaufortia multiocellata Nguyen & Nguyen, 2005: 590-592. Beaufortia triocellata Nguyen & Nguyen, 2005: 592-594. Beaufortia zebroida:. Kottelat (2012): 59-60. zse.pensoft.net Comparative materials. MMNHN1544, one specimen, holotype, not found yet, refers to the original descrip- tion. SHOU20240111301-317, 33.24-49.96 mm SL, 17 specimens from Guangnan County, Xichou County, Ma- lipo County, Funing County, Yunnan, and Napo County, Guangxi, SHOU20240111301-317, 33.24-49.96 mm SL, were collected by Lao Xing and Lin Yang from Decem- ber 2021 to January 2024. Diagnosis. B. zebroida shares the typical character- istics with members of the B. pingi species group, with distinct vertical stripes on lateral body and a pinnate-type lower lip (vs. lacking prominent vertical stripes and having a dicot-type lower lip in other congeneric species apart from this group) (see Figs 6B, 7, Table 2). B. ze- broida can be clearly distinguished from B. granulopin- na sp. nov. by the absence of prominent tubercles on the pectoral fin rays (vs. well-developed tubercles present on anterior 6—9 rays of pectoral fins); vertical stripes slender, narrower than inter-spacing, present consistent- ly at all growth stages (vs. vertical stripes typically wid- er than inter-spacing, with a certain proportion (54.76%) of adults exhibiting blurred or vanished mid-section stripes). B. zebroida can be clearly distinguished from B. viridis sp. nov. by the vertical stripes present uneven in width and length, typically narrower than inter-spac- ing, sometimes reduced to dots (vs. stripes of consistent length, width, and inter-spacing); body brown yellow to golden in dorsal profile (vs. dark cyan to green). B. ze- broida can be clearly distinguished from B. pingi by the stable presence of vertical stripes at all growth stages (vs. a large proportion (94.00%) of adults with blurred or vanished vertical stripes at mid-section of lateral body) and body brown yellow to golden in dorsal profile (vs. dark brown to green). Redescription. Dorsal 11-7; anal 1-3-4 (3.98+0.14); pectoral 1-20—23 (21.5+0.73); pelvic 1-16-19 (17.53+0.86). Lateral-line canal pores and scales: 70-84 (75.12+3.55). Zoosyst. Evol. 100 (3) 2024, 941-963 AAs SS » ).)H C= SS Guangnan County, Yunnan 9od Napo County, Guangxi Figure 15. Lateral (top), dorsal (middle), and ventral (bottom) views of different regional phenotypes of Beaufortia zebroida. A. Regional phenotype from a stream tributary to the Red River, Guangnan County, Yunnan, SHOU20240111308, 36.64 mm SL; B. Regional phenotype from Napo County, Guangxi, SHOU20240316602, 56.12 mm SL, originating from upstream within the same basin as the type locality. Morphometric measurements for the specimens exam- ined are given in Table 3. See Fig. 15 for lateral, dorsal, and ventral views of the body. Body closely resembles B. viridis sp. nov. in general shape and structure, but significantly different in stripes pattern and coloration. Coloration in preservation. Preserved specimens from sub-adult to adult stage, body brown yellow to grey, white ventrally. Head with black spots or vermiculation dorsally, 2—5 larger black blotches along mid-dorsal body anterior to dorsal fin. Sides with 8—15 vertical dark stripes, varying greatly in length, which sometimes ap- pearing as dots, stripe width narrower than inter-spacing, present at all growth stages. Paired fin margins hyaline or pale white, inner edges with continuous or dotted black arc. Dorsal fin hyaline, with black stripes, one black spot on the root before the second branched ray. Coloration in live. In life, body brown yellow to gold- en in dorsal profile. Mature individuals with a more vivid golden coloration, and fin membranes pale yellow. Individual variation. Among 17 specimens, one (SHOU20240111307) had three branched anal fin rays. Ethology. Inhabits shallow streams with rapid currents and smooth pebble substrates that adhere to crevices be- tween stones. Feeds on algae and small invertebrates and consumes mucus from fresh fish carcasses. Exhibits ter- ritorial behavior. Distribution. Exclusively in small tributaries on the north bank of the mid-lower Red River system and the upper Shuikou River to Zuo River basin (upper Pearl River system), from Maguan County, Wenshan Zhuang, and Miao Autonomous Prefecture, Yunnan, to Longzhou County, Guangxi, near the China-Vietnam border, ex- tending into northeastern Vietnam (see Fig. 9). Genetic distances and phylogenetic analysis A total of 35 mitochondrial cytb gene haplotypes were obtained. The computed genetic distances revealed that B. granulopinna sp. nov. shared the smallest average genetic distance with B. zebroida at 10.80% (with the maximum interspecific genetic distance reaching 11.37%). Intraspe- cific genetic distances ranged from 0.26% to 5.12%, with an average of 3.63%, surpassing the average intraspecific genetic distances of all other species within the same ge- nus. B. viridis sp. nov. exhibited a minimum average in- terspecific genetic distance with B. pingi of 4.60% (with a maximum interspecific genetic distance of 5.2%), signifi- cantly exceeding the current minimum intrageneric inter- specific distance. Intraspecific genetic distances ranged from 0.09% to 2.80%, with an average of 1.47%, exceed- ing the average intraspecific genetic distances of all other congeneric species except for B. granulopinna sp. nov. B. zebroida had a minimum average genetic distance with B. pingi of 5.62% (with the maximum interspecific ge- netic distance at 5.88%), considerably above the current minimum intrageneric interspecific distance. The genetic distances between the aforementioned species and those within the B. pingi species group were smaller, indicating zse.pensoft.net 958 their affiliation with the B. pingi species group, aligning with morphological classification results. Notably, the genetic distance between B. niulanensis and B. szechuanensis was only 0.53%, whereas the B. cf. szechuanensis collected from Quijiang, Chongqing (a trib- utary on the southern bank of the upper Yangtze River) morphologically conformed to B. szechuanensis but ex- hibited a genetic distance of 2.30% from B. szechuanen- sis, exceeding the genetic distance between B. polylepis and B. szechuanensis (2.20%) (see Table 5). Phylogenetic trees generated using maximum likelihood and Bayesian inference methods exhibited identical topo- logical structures, with all nodes demonstrating high support values. All species within the B. pingi species group co- alesced to form a single clade, with each species within this Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group clade establishing distinct lineages. Among these, B. pingi and B. viridis sp. nov. were identified as sister taxa posi- tioned at the derived end of the phylogenetic tree, followed by B. zebroida as a sister group to the aforementioned spe- cies. B. granulopinna sp. nov. was situated at the most basal position within the B. pingi species group clade, with a rel- atively longer branch length indicating an early divergence. The remaining species formed another clade, distinctly divergent from the B. pingi species group, which aligns with the classification results based on morphological characteristics. Within this clade, two significantly diver- gent lineages were identified. One lineage was composed of all haplotypes of B. kweichowensis and B. leveretti. The other lineages included B. szechuanensis, B. polylepis, B. niulanensis, and B. cf. szechuanensis. (see Fig. 16). Table 5. Inter-specific mean mitochondrial cyfb genetic distances, maximum genetic distances among species, and intra-specific genetic distances within the genus Beaufortia in Kimura 2-parameter genetic distance analysis. Species (haplotype count) 1 2 3 4 1B. viridis sp. nov. (5) 5.20 S20 (11 AS 2B. pingi (8) 4.60 5.88 11.46 3B. zebroida (4) 7.07 5.62 11.37 4B. granulopinna sp. nov. (5) 10.86 11.15 10.80 5B. kweichowensis (3) 153% lisse 1c66r 163 6B. leveretti (1) T:51« eto. Tos0« “1625 7B. niulanensis (2) 15.77. 16.92 16.05 17.04 8B. szechuanensis (2) 16.16 17.25 16.31 17.39 9B. polylepis (4) 16.28 1684 15.84 16.94 10 8B. cf. szechuanensis (1) 16.44 17.54 16.16 17.37 (eb) stands for no corresponding data. 0.1 96/99 91/* 99/* Between species mean distance (below diagonal) and maximum distance (above PP482583 Qujing City eameg Coe y PP482584 Quijing City NC027291 PP482569 Neijiang City PP482570 Kaiyuan City PP482572 Luoping County PP482594 Luoping County PP482571 Shizong County Ae See o PP482573 Qijiang District sea fran aie wee iar rs: Brenner oes 8 PP482574 Congjiang County PP482576 Lingchuan County PP482575 Huizhou City PP482577 Baisha County KF732713 Within species diagonal)) (%) distance (%) 5 6 7 8 9 10 Range Mean 15.71 15.74 17.40 17.67 17.54 17..83 0.09-2.80 1.47 15.70 16.33 17.37 17.77) 17.26 17.93 0.09-0.89 0.50 16.29 15.56 16.27 1666 16.04 16.31 0.09-0.53 0.28 16.43 16.97 17.78 17.51 17.18 17.67 0.26-5.12 3.63 3.86 14.22 1400 14.24 14.02 0.09-2.06 137, 3.44 13.64 13.65 13.89 13.80 - - 13.80 13.58 0.71 2.34 Vaeis) 0.09 0.09 13.71 13.53 0.53 2.43 2.53 0.44 0.44 PGT: TiS Ake, Ale. 2.20 3.09 0.18-0.35 0.23 13782 nl 3:80" 2.20 2.30 2.90 - - B. pingi species Group | Beaufortia niulanensis | Beaufortia szechuanensis Beaufortia polylepis i Beaufortia cf. szechuanensis Beaufortia kweichowensis | Beaufortia leveretti | Plesiomyzon baotingensis (outgroup) Figure 16. Phylogenetic tree of Beaufortia species based on haplotypes of mitochondrial cytb genes, with the tree topology inferred through maximum likelihood (ML) and Bayesian inference (BI) methods. Support values for each branch are indicated at the nodes (ML/BI), with “*” denoting bootstrap values of 100% or posterior probabilities of 1, and “-” indicating insufficient genetic variation to display posterior probabilities. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 941-963 Morphological characteristics of the Beaufortia pingi species group The preceding phylogenetic results have confirmed that the Beaufortia pingi species group constitutes a mono- phyletic clade and exhibits significant genetic diver- gence from other Beaufortia species. Morphologically, this group is distinctly differentiated from other species. The lower lip of the species group can be represented by a pinnate-type appearance, being overall slender with a central indentation and multiple lobed sides. There is variation among individuals in the number of lobes and the depth of the clefts, with most specimens possess- ing no fewer than three lobes and the depth of clefts exceeding that found in species outside this group. The extremities of the lower lip extend to the corners of the mouth, with several wart-like protrusions present on the extended parts (Fig. 6). In contrast, species outside this group possess a dicot-type lower lip, which is broad and short with a central indentation, and sides that are not lobed but occasionally have 0-2 shallow notches. The ends of the lower lip do not extend to the corners of the mouth, or the extension is poorly developed and lacks protrusions (Fig. 7). Additionally, the Beaufortia pingi species group 1s characterized by distinct vertical stripes along the body sides, a feature absent in other Beaufortia species. Morphometric variability within the Beaufortia pingi species group B. pingi has more branched rays in its paired fins, with an average of 22.19+0.78 in pectoral fins and 18.64+0.97 in pelvic fins. In contrast, B. granulopinna sp. nov. ex- hibits fewer branched rays in its paired fins, averaging 20.11+0.77 in pectoral fins and 16.51+0.86 in pelvic fins. Differences in morphometric proportions were ob- served among the four species within the Beaufortia pingi group. B. granulopinna sp. nov. and B. pingi have comparatively taller bodies, as reflected in lower mean SL/BD ratios (5.1040.43 and 5.09+40.71, respectively) compared to those observed in B. viridis sp. nov. and B. zebroida (5.31+40.54 and 5.47£0.33, respectively). B. pingi is distinguished by a broader body, as evidenced by smaller mean ratios of SL/BW and SL/PW (4.2+0.36 and 7.42+0.39, respectively), a shorter head, indicated by a larger mean SL/HL (4.9+0.18), and a narrower mouth, denoted by a larger mean HW/MW (5.79+0.5). Com- pared to the other three species, B. granulopinna sp. nov. features a shorter and thicker caudal peduncle, as shown by the smaller mean SL/CD and PoAL/CD (8.36+0.52 and 1.29+0.11, respectively). Variability in morphometric characteristics is ob- served within populations of B. viridis sp. nov. from different regions. Specimens from Lingyun County possess a more robust anterior body segment, with wid- er and taller heads and trunks (PrPvL/HW=1.53+0.08, 959 PrPvL/PW=0.99+0.03, PrPvL/BW=1.73+0.07, PrPvL/ BD=1.98+0.15, PrPvL/HD=2.48+0. 12, PrPvL/ PW=1.25+0.05), and a thicker caudal peduncle (PrPvL/ CD=3.59+0.28). The population from the type locality, Wuming District, Nanning City, exhibits fewer branched rays in the paired fins (pectoral fins 20.25+0.68, pelvic hns16:5+0371) Compared to the type locality, the population from De- bao County is characterized by a broader and flatter body (PW/BD=2.32+0.19) and a thinner caudal part (PoAL/ CD=1.69+40.09) (see Table 4). It is noteworthy that, despite the statistically signifi- cant and visually discernible differences in measurable characteristics, the presence of overlap among different species precludes recommending these characteristics as reliable bases for species identification. Discussion Taxonomic clarification and redescription of Beaufortia pingi In 1930, Fang described Gastromyzon pingi with the type locality specified as Lingyun County in 1928. He mentioned two collection sites, one near the boundary of Yunnan Province (type locality) and the other in Dama- oping Village. At that time, the jurisdiction of Lingyun County was more extensive, bordering Yunnan and en- compassing the current eastern area of Tianlin County. The county’s watershed includes the Xiyang River, Bo'ai River, Jia River (currently Leli River), and Chengbi Riv- er. This broad and somewhat vague collection range coin- cides with the primary distribution area of the Beaufortia pingi species group. Given the relatively short geograph- ical distances between distribution areas of different species within this region, it is highly probable that the type specimens could actually belong to different species. The paratype specimen NO. 955 exhibits a notably lower count of pectoral fin rays, a distinguishing characteris- tic of B. granulopinna sp. nov. from B. pingi. Excluding the lateral line scale count, the morphological features of the remaining specimens align with those of B. pingi specimens collected from the Chengbi River basin within the current boundaries of Lingyun County. The scales of the B. pingi species group are exceptionally fine, often less than 0.5 mm in diameter, suggesting that the original counts might have been inaccurately high due to the lim- itations of observation equipment. Apart from the Chengbi River basin, B. pingi was also collected from the Jian River basin in Debao County. Compared to the holotype, these specimens have slightly fewer ventral fin rays and a somewhat broader and flatter body, but they share the main differential features of the species. The genetic distance of the mitochondrial cytb gene is less than 1%, indicating minor genetic variation, and phylogenetically, these specimens cluster within the same lineage as B. pingi. zse.pensoft.net 960 In summary, the species within the Beaufortia pingi group are geographically proximate and morphologically similar. The type locality of B. pingi is relatively indis- tinct, and the age of the specimens complicates identifica- tion. The inclusion of other species among the paratypes, the original vague description, and some inaccuracies in detail present challenges. Considering these issues, we have conducted a redescription of B. pingi based on ex- tensive specimen collection, morphological comparisons, and molecular phylogenetic analysis. Taxonomic clarification and redescription of Beaufortia zebroida Gastromyzon pingi zebroidus was also described in the same article, in which Fang (1930) described Gastro- myzon pingi with a single-type specimen collected from the Dongui River along the China-Vietnam border in Longzhou County, Guangxi Province. Since its publica- tion, no further records of this species have been found in that basin. Despite multiple surveys in Longzhou County, the species was not rediscovered, and the local rivers, mostly altered by human activities and facing severe bioinvasions, significantly reduced the habitats suitable for gastromyzonids. The limited information from a single specimen, cou- pled with the low distinctiveness of the illustrations and the relatively vague morphological description of Gastro- myzon pingi zebroidus, has led to significant controversy over the identity of this species and multiple changes in its taxonomic status. In the original description, Fang (1930) distinguished this subspecies by the anal pore’s proximity to the pos- terior edge of the ventral fins and the presence of stripes along the entire flank (vs. the anal position being further from the posterior edge of the ventral disc and stripes being pronounced at the caudal peduncle in Gastromy- zon pingi). Later, Hora (1932) elevated its taxonomic status to species level as Beaufortia zebroidus with- out providing justification. Nichols (1943) followed Fang’s (1930) perspective, considering it a subspecies of Gastromyzon pingi. Chen (1980), upon examination of two specimens from the Xijiang River (location unspecified), with total lengths of 29 mm and 52 mm, respectively, observed that both specimens exhibited pronounced or faint vertical stripes and noted the anal pore situated at a considerable distance from the ventral fins. Consequently, Chen tenta- tively synonymized Beaufortia zebroidus with Beaufortia pingi, pending further collection of specimens for a defin- itive conclusion. According to our observations, the ver- tical stripes on juvenile stages of B. pingi typically do not disappear, and the specimen measuring 29 mm in length is likely in a late juvenile stage. Therefore, the species in question is inferred to be B. pingi. Yue (1981), upon examination of nine specimens from Lingyun County, Guangxi, identified them as B. pingi and zse.pensoft.net Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group noted the presence of 2-4 transverse vertical stripes on the caudal peduncle. The presence and clarity of stripes anterior to the caudal peduncle varied among individuals, with some exhibiting up to 10 distinct stripes. The anal pore was observed to be closer to the anal fins than to the posterior edge of the ventral fins, with distance varying across the specimens. Notably, one specimen, No.74664, possessed an anal pore precisely at the posterior edge of the ventral fins. Based on these findings, Yue proposed that B. zebroidus could be a synonym of B. pingi or not a valid subspecies. Zheng (1989) examined 15 specimens from Lingyun County, Guangxi, and Xingyi County, Guizhou, and identified them as B. pingi. Zheng observed the terminal rays of the ventral fins connecting medially on both sides, with a notch at the end (presumably a ty- pographical error for “a pair of notches”), the posterior edges located near the anal pore or slightly distant, and 11-13 transverse bands across the dorsal midline accom- panied by approximately 20 vertical stripes. The presence of unclear stripes, visible only at the caudal peduncle in some individuals, led Zheng to consider B. zebroidus a ju- nior synonym of B. pingi. Both researchers examined the specimens from the type locality, and given that Lingyun County is habitat to both B. pingi and B. viridis sp. nov., the possibility of these specimens comprising two distinct species cannot be excluded. Subsequently, Chen (1990) examined 21 specimens from the Xiyang River in Guangnan County, Yunnan, identifying them as B. pingi. The observations revealed that the tips of the pelvic fin rays were proximal to the anal pore in all specimens, with numerous dark and light stripes along the dorsal midline and flanks. Chen thus regarded B. zebroidus as a junior synonym of B. pingi. Our research indicates that the species distributed in the Xiyang River is B. viridis sp. nov., and given the con- sistent presence of vertical stripes in all specimens, the referenced species 1s likely B. viridis sp. nov. Later, Chen and Tang (2000) measured three spec- imens from Tian’e, Guangxi, noting that the posteri- or edges of the pelvic fins were proximal to the anal pore. The dorsal and lateral sides exhibited approxi- mately 20 black vertical stripes, with the pre-pelvic length significantly exceeding the distance from the pelvic fin origin to the anal fin origin. The branched ray count in the paired fins (pectoral 19, pelvic 15-16) was slightly lower than that observed in the specimens from the type locality of B. pingi (pectoral 21-23, pelvic 18-20), leading them to recognize B. zebroi- dus as a valid species. We noted that the branched ray counts in the paired fins of the specimens collect- ed from Tian’e County fell within the variation range of the specimens from the type locality of B. viridis sp. nov., with the pre-pelvic length generally greater than the length from the pelvic origin to the anal ori- gin (mean PrPvL/POAO=1.19+0.74), distinct from the specimens from the type locality of B. pingi (PrPvL/ POAO=0.98+0.66). Hence, the specimens collected from Tian’e County are identified as B. viridis sp. nov. Zoosyst. Evol. 100 (3) 2024, 941-963 Chen and Zhang (2006), after examining six spec- imens from Tianlin County and Lingyun County and referencing Chen and Tang (2000), encountered individ- uals exhibiting characteristics of both B. pingi and B. ze- broidus (presence or absence of vertical stripes, count of branched rays in paired fins, PrPvL/POAO ratio), thereby considering B. zebroidus a synonym of B. pingi. Tianlin County serves as the type locality for B. granulopinna sp. nov. Our statistical analysis indicates that a certain proportion of vertical stripes disappear in the adult stages of both B. granulopinna sp. nov. and B. pingi (54.76% and 94.00%, respectively). The branched ray counts in the paired fins of B. granulopinna sp. nov. are also mar- ginally less than those of B. pingi, and the PrPvL/POAO ratios for both species fluctuate around 100% (as shown in Table 3). Moreover, we observed that the photographs provided by the authors (original fig. X 98), which de- picted B. granulopinna sp. nov., showed the first few rays of the pectoral fins exhibiting well-developed tubercles. Therefore, the batch of specimens actually represents a mix of B. pingi and B. granulopinna sp. nov. from their type localities. Finally, Kottelat (2012) recognized B. zebroidus as a valid species, changing the suffix to -a, and synonymized B. fasciolata, B. multiocellata, and B. triocellata, collect- ed from the China- Vietnam border, with B. zebroida. After investigating all possible distribution areas and combining morphological analysis with mitochon- drial cytb gene phylogenetic analysis, we conclude the following: Key to the Beaufortia pingi species group 961 The type locality of B. zebroida is the border river be- tween China and Vietnam. Among all the literature re- cords, the ones closest to the type locality are the collection sites of B. fasciolata, B. multiocellata, and B. triocellata. Although the type specimens of these three species are lost, their original descriptions and unclear illustrations still confirm that they belong to the same species as our specimens collected from the southeastern Yunnan Prov- ince to the western Guangxi border area with Vietnam. Fang (1930) mentioned the specimen color as pale fleshy, which aligns with the body color of the aforementioned populations but not with B. pingi and B. viridis sp. nov. Thus, we consider B. zebroida a species distributed from Maguan County in Yunnan Province to Longzhou Coun- ty in Guangxi Province along the China- Vietnam border, as well as in northeastern Vietnam. The region contains small tributaries flowing to both the Red River and the Zuo River (Pearl River system), closely spaced due to the karst landscape, facilitating stream capture and exchange. The type locality, Longzhou County, represents the east- ern boundary of the species’ distribution, but due to habi- tat destruction and bioinvasions, no further records of this species have been found there. Besides this area, the spe- cies has been discovered in Maguan County, Guangnan County, Xichou County, Malipo County, Funing County, and Napo County (stream tributary to the upper reach- es of the Shuikou River, same basin as the type locality) within China and northeastern Vietnam. Following the clarification of its taxonomic status, we have provided a redescription of B. zebroida. 1 Vertical black stripe on flank absent; lower lip dicot-type, sides not lobed or with 1-2 small notches...other Beaufortia species — Flank with multiple vertical dark stripes; lower lip pinnate-type, SIDES MUITIHODE 2.0... cece cecc eee eecee eee eeeeeeeeeeeeeneeaeeees 2 2 Adult pectoral fins with 6-9 rays bearing well-developed granular nuptial tubercles ...............::ceceeeeeeeeee ees B. granulopinna =4 eNuptial tubercles: onracullépecteral Titrays. do Sen Og MIGIStINCly neko tty TA dics Ga Ae seeing la ae 2 3. Vertical stripes in a large proportion (94.00%) of adults show blurred or vanished before caudal peduncle ........ B. pingi -— Vertical stripes consistently present at all developmental stages, not blurring OF VANISHING ........... cece eee eeee eee eeeeeeeeeeneenes 4 4 Vertical stripes uniform in width and length, often wider than spacing; dorsal body dark cyan to green............... B.viridis Stripes uneven in width and length, often narrower than spacing, sometimes reduced to dots; body brown yellow to SOMO, CHONS El cs Pe cert ne IS, Bere Ve WB em 5 er ee bee Oe ree Ce AA es cons 03 PPT ae tres in Be oo er ed eda ee oe or B.zebroida Survival status and conservation recommendations for the Beaufortia pingi Species group The research on the Beaufortia pingi group is mostly in its initial stages; however, their situation is far from optimis- tic. These species have become popular ornamental fish 1n China. Aquarium trade operators reap substantial profits through the capture and sale of these fish from the wild, yet among them are those who act with disregard for sus- tainability; their harvesting practices are often destructive. Given their rheophilic nature, projects that obstruct rivers can easily lead to regional extinction. Their low pollution tolerance and sensitivity to changes in water quality also contribute to the significant reductions in population that many species are suffering. In the type locality of B. vir- idis sp. nov., Wuming District, Nanning City, most small tributaries have been modified into step-like reservoirs for water storage, and those near agricultural irrigation areas are polluted, rendering these areas unsuitable for their survival. Interestingly, a stable population was dis- covered inside a commercial eco-camping site. To satisfy consumers’ pursuit of “pristine nature,” contractors have left some river sections poor developed, providing a ref- uge for this species. We suggest that future efforts should focus on increasing attention to these species, conducting zse.pensoft.net 962 fundamental research, and further exploring their scien- tific and economic potential. Simultaneously, it is crucial to enhance habitat conservation awareness, scientifically plan, and develop sustainably, ensuring harmonious co- existence between humans and nature. Comparative materials Beaufortia szechuanensis: SHOU20231220601, one specimen, 59.18 mm SL, from Wanzhou Dis- trict, Chongqing, upper Yangtze River basin. SHOU20231220602, one specimen, 42.94 mm SL, from Qijiang District, Chongqing, upper Yangtze Riv- er basin. SHOU20231220640-642, three specimens, 46.17-54.93 mm SL, from Neijiang City, Sichuan, up- per Yangtze River basin. Collected by Jing-Chen Chen and Hao-Tian Lei. Beaufortia liui. SHOQU20240225701, one specimen, 36.64 mm SL, from Huidong County, Sichuan, upper Yangtze River basin. Collected by Can Liu. IHAS- W83VI0424-425, two specimens, from Huidong County, Sichuan, upper Yangtze River basin, inspec- tion only. IHW 2247, 2446, two specimens, syntype, inspection only. Beaufortia _niulanensis: SHOU20231220614-615, SHOU20231220617, three specimens, 41.38—-43.66 mm SL, from type locality, Qujing City, Yunnan, upper Yangtze River basin. Yangtze River basin. Collected by Xin-Rui Pu, Lao Xing, and Lingzi. Beaufortia polylepis: SHOU20231220643-644, two spec- imens, 40.06—41.02 mm SL, from downstream near the type locality, Karyuan City, Yunnan, upper Nanpan River (Pearl River system). Collected by Lin Yang. IHW 774330, one specimen, syntype, inspection only. Beaufortia huangguoshuensis. THASW83IV0049, one specimen, syntype, and fin rays were counted; the oth- er data refers to Chen and Tang (2000). Beaufortia intermedia: IHW 87IV516, one specimen, paratype, inspection only. Measurement data refers to Tang et al. (1997). Beaufortia leveretti: SHOU20231212626-627, two specimens, 44.65-52.42 mm, from Fangcheng- gang City, Guangxi, collated by Quian-Yu Liang. SHOU20231212628-630, three specimens, 59.94— 64.71 mm, from type locality, Baisha County, Hainan, collector unknown. Beaufortia kweichowensis. SHOU20231212601-602, two specimens, 53.77-55.24 mm, from Congjiang County, Guizhou, Duliu River (Pearl River system). SHOU20231212614-616, three specimens, 44.38— 47.77 mm, from Pingle County, Guangxi, Li River (Pearl River system). Collected by Jing-Chen Chen. Beaufortia cyclica: T\HASW75IV 1417, one specimen, ho- lotype, inspection only. Measurement data refers to the original description. Beaufortia yunnanensis, Beaufortia fasciolata, Beaufortia triocellata, Beaufortia multiocellata, Beaufortia buas, zse.pensoft.net Chen, J.-C. et al.: Taxonomic resolution of Beaufortia pingi species group Beaufortia daon, Beaufortia elongata, and Beaufortia loos. Referenced original descriptions (Li et al. 1998; Kottelat 2001; Nguyen and Nguyen 2005). Contributions Jing-Chen Chen conducted sample collection, speci- men identification and measurement, photography, map drafting, molecular genetic analysis, and manuscript composition. Jia-Jia Li was responsible for specimen measurement, illustration of measurement methodolo- gies, and molecular genetic experimentation. Wen-Qiao Tang provided guidance on research concepts as well as review and revision of the manuscript. Xin-Rui Pu par- ticipated in sample collection and specimen identifica- tion. Hao-Tian Lei carried out specimen collection and grammatical checks. Acknowledgments We sincerely thank the Institute of Hydrobiology, Chinese Academy of Sciences, for their assistance in the inspec- tion of type specimens. Our deep appreciation goes to Mr. Jia-Jun Zhou, Mr. Qian-Yu Liang, Mr. Lao Xing, Mr. Lin Yang, Mr. Guo Yu, Mr. X1-Xian Chen, and Mr. Can Liu for their invaluable support and assistance with sample collection. We also extend our thanks to Yi- Yang Xu and Hao-Yang Xie for their contributions to photographing the type specimens. Special thanks are due to Zhi-Xian Sun for his guidance and assistance with photography. We are grateful to the Library of Nha Trang Universi- ty for providing the necessary reference materials. This work was supported by the National Natural Science Foundation of China (NSFC) (Grant No. 31093430) and the National Special Program for Basic Research Works in Science and Technology (2015FY110200). References Chen YY (1980) Systematic studies on the fishes of the family Hom- alopteridae of China II. 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