Zoosyst. Evol. 100 (2) 2024, 663-673 | DOI 10.3897/zse.100.118061 Gag MuseuM TOR BERLIN Taxonomic revision of the cavefish genus Karstsinnectes (Cypriniformes, Nemacheilidae), with a description of a new species from Guangxi Province, China Jia-Yue Ge! 2", Zheng-Quan Nong*", Jian Yang*, Li-Na Du? , Jia-Jun Zhou?’® 1 Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, Guilin, Guangxi 541004, China 2 Guangxi Key Laboratory of Rare and Endangered Animal Ecology, College of Life Science, Guangxi Normal University, Guilin, Guangxi 541004, China Administration Center of Guangxi Nonggang National Nature Reserve, Longzhou Guangxi 532400, China Key Laboratory of Environment Change and Resource Use, Beibu Gulf, Nanning Normal University, Nanning, Guangxi, 530001, China Zhejiang Forest Resource Monitoring Center, Hangzhou, Zhejiang 310020, China Dn oO fF W Zhejiang Forestry Survey Planning and Design Company Limited, Hangzhou 310020, China https://zoobank. org/0440F 4DE-BECE-4B8B-9D8&4-S8E 17489226C Corresponding authors: Li-Na Du (dulina@mailbox.gxnu.edu.cn); Jia-Jun Zhou (cnwaters@foxmail.com) Academic editor: Nicolas Hubert # Received | January 2024 Accepted 17 April 2024 Published 27 May 2024 Abstract The blind cavefish genus Karstsinnectes, established in 2023, is the subject of taxonomic revision in the present study. Five valid species are recognized, including one new species, Karstsinnectes longzhouensis sp. nov., described from Guangxi, China, based on a combination of morphological evidence. Karstsinnectes longzhouensis sp. nov. can be distinguished from all other congeners based on the presence of a lateral line, 11-12 branched pectoral fin rays, and five branched pelvic fin rays. Additionally, due to the loss of the type specimens, a neotype is designated for K. parvus. The lateral line of K. hyalinus is revised as lacking in this study. A key to all valid Karstsinnectes species is provided. Blind cavefish serve as a valuable natural framework for investigating con- vergent and adaptive evolutionary processes. The survival of cavefish is under significant threat due to human activities, climate change, water pollution, and invasive species. Thus, to preserve these valuable species, it is crucial to implement various conserva- tion measures, such as habitat protection, artificial breeding, and fundamental research. Key Words Blind cavefish, complete mitochondrial genome, morphology, Taxonomy, Xijiang River Introduction Karst caves and subterranean streams represent domi- nant geomorphological features in the Guangxi, Guizhou, Sichuan, and Yunnan provinces and Chongqing City of China. These regions are renowned for harboring many unique cave-dwelling fish species (Zhao and Zhang * These authors contributed equally to this work. 2009). Ma et al. (2019) recorded 148 hypogean species in China, including 65 nemacheilids in Heminoemachei- lus, Oreonectes, Protocobitis, Paranemachilus, Schistu- ra, Troglonectes, Triplophysa, and Yunnanilus. Luo et al. (2023) described a new genus, Karstsinnectes Zhou, Luo, Wang, Zhou & Xiao, 2023, with Oreonectes anophthal- mus Zheng, 1981, as the type specimen, based on morpho- Copyright Ge, J.-Y. et a/. 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. 664 logical characters and molecular evidence. The diagnostic characters of Karstsinnectes include body scaleless and colorless, anterior and posterior nostrils separated, the base of the anterior nostril tube-shaped, a tip not elongat- ed to barbel-like, adipose crests present on the caudal pe- duncle, lateral line and cephalic lateral line canals present, and the body capsule of the swim bladder open posteriorly (Luo et al. 2023). Four species within the genus Karstsin- nectes have been recorded, including K. acridorsalis (Lan, 2013), K. anophthalmus (Zheng, 1981), K. hyalinus (Lan, Yang & Chen, 1996), and K. parvus (Zhu & Zhu, 2014). In December 2022, four Karstsinnectes specimens were collected from a cave in Longzhou County, Chongzuo City, Guangxi, China. Morphological characters and mo- lecular analyses indicated that these specimens represent- ed an undescribed species of Karstsinnectes. Additionally, a taxonomic revision of K. acridorsalis and K. hyalinus is provided, and a neotype is designated for K. parvus herein. Materials and methods All care and use of experimental animals complied with the relevant laws of the Chinese Laboratory of Animal Welfare and Ethics (GB/T 35892-2018). Specimens of Karstsinnectes longzhouensis sp. nov. were rapidly euth- anized by an overdose of anesthetic clove oil. The right- side pectoral fin and pelvic fin were excised and preserved in 99% ethanol. Specimens for morphological study were initially stored in 10% formalin, then transferred to 75% alcohol for long-term preservation at the Kunming Nat- ural History Museum of Zoology, Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). Counts and measurements followed Du et al. (2021) and Luo et al. (2023). Data were initially processed us- ing Microsoft Excel software for preliminary statistical analysis. The original data were converted to percentag- es of standard length (SL) or lateral head length (HL), followed by logarithmic conversion (log10) to remove the effects of allometry. Comparative morphometry was examined using principal component analysis (PCA) in IBM SPSS Statistics v20.0. Non-parametric analysis of variables with principal component loadings greater than 60% was used to determine the degree of differences be- tween subjects, and the scores of each principal compo- nent were used to generate scatter plots for analysis. Complete mitochondrial genome sequencing data were submitted to GenBank under Accession No. OR947935. The mitogenome of OR947935 was sequenced on I/lumi- na Novaseq 6000 (Origingene Bio-pharm Technology Co. Ltd., Shanghai, China). The quality of sequencing raw data was evaluated by Fastqc (v.0.11.8) and trimmed using Cu- tatapt (v.4.8) software (Martin 2011). Obtained Illumina reads were de novo assembled using the NOVOPlasty soft- ware (Dierckxsens et al. 2017), and the assembled genome was annotated by Mitos2 (Bernt et al. 2013). Relative syn- onymous codon usage (RSCU) in protein coding sequenc- es and simple sequence repeats (SSRs) of OR947935 were determined in CodonW (v.1.4.2) and MISA, respective- zse.pensoft.net Ge, J.-Y. et al.: A taxonomic revision of Karstsinnectes ly (Peden 1999; Beier et al. 2017). To test the phyloge- netic position of Karstsinnectes longzhouensis sp. nov., Bayesian inference (BI) was performed using MrBayes in XSEDE (v3.2.7a) by CIPRES Science Gateway (Mill- er et al. 2010). The entire sequence of the mitochondrial genomes was used as a single partition. The substitution model GTR + I + G was selected as the best model us- ing jJModelTest v.2.1.10 (Darriba et al. 2012). Likelihood Model parameters were set as number of substitution types allow all rates to be different, subject to the constraint of time-reversibility (Nst = 6), nucleotide substitution mod- el. Nucmodel was standard model of DNA substitution in which there are only four states (Nucmodel = 4~4), the rate at a site is drawn from a gamma distribution, and all site patterns had the possibility of being sampled (Coding = all). Forty complete mitochondrial genomes obtained from GenBank were included in the data. Parabotia fasciata Dabry de Thiersant, 1872 and Leptobotia elongata (Bleek- er, 1870), two botiid species, were used as outgroups. Two runs were performed simultaneously with four Markov chains starting from a random tree. The chains were run for five million generations and sampled every 100 gen- erations. The first 25% of sampled trees were discarded as burn-in, and the remaining trees were used to create a con- sensus tree and estimate Bayesian posterior probabilities (BPPs). Uncorrected pairwise distances between species of Karstsinnectes were calculated in MEGA v11. Results Genus Karstsinnectes Zhou, Luo, Wang, Zhou & Xiao, 2023 Karstsinnectes Zhou, Luo, Wang, Zhou & Xiao, 2023, 696 original de- scriptions. Type species. Oreonectes anophthalmus Zheng, 1981. Diagnosis. Base of anterior nostril tube-like and tip not elongated to barbel-like; anterior and posterior nostrils adjacent; lips with furrows; caudal peduncle with adipose crests; bony capsule of swim bladder opens posteriorly. Remarks. In the family Nemacheilidae, the genera Eo- nemachilus Berg, 1938; Karstsinnectes; Micronemachei- Jus Rendahl, 1944; Protonemacheilus Yang & Chu, 1990; Traccatichthys Freyhof & Serov, 2001; and Yunnanilus Nichols, 1925, are characterized by a tube-like anterior nostril with tip not elongated to barbel-like structure (Du et al. 2021, 2023). However, Karstsinnectes can be distin- guished from Eonemachilus, Protonemacheilus, Tracca- tichthys, and Yunnanilus by anterior and posterior nostrils adjacent (vs. separated in Eonemachilus and Yunnanilus, closely set in Protonemacheilus and Traccatichthys) and from Micronemacheilus and Traccatichthys by lips with furrows (vs. with papillae). Species included. Karstsinnectes acridorsalis (Lan, 2013), K. anophthalmus (Zheng, 1981), K. hyalinus (Lan, Yang & Chen, 1996), K. longzhouensis sp. nov., and K. parvus (Zhu & Zhu, 2014). Zoosyst. Evol. 100 (2) 2024, 663-673 Key to species of Karstsinnectes 1 a Clall, MMCRO MCAS ect. = nacS Se hc oasss oonediedyseteeccs:itesass SaiCalanifitOEKe Ct. |22.:% enim: tance rete te el ee eee aos teeth Body covered by SCaleSi bracers 2a ent beer Ane - BOY SCALSIESS Se cms ris. a Eee operate AN PORERS orn ers LS 3 Lateral: (ier Ore Se iia: : tmtee once Minette eae enact anaes ctantee - Kateral lier bSemte. 82 cee sll ii eee NM | iN 665 ie Bs ER CR ey bk: a ear eee kK. anophthalmus SD Sst RR REE Ss 2 ME Oy pad tS ES EE K. hyalinus Bernie tll rei nN el Pel ERS ee IE le ronan LAL, K. acridorsalis Pecterall Tiwith- lO prayenearays, TiVe bFanehegspelwicHiaray Sy ees oss s5c Poh eed does car ners US ete K. parvus Pectoral fin with 11 or 12 branched rays, six branched pelvic fin rayS............cceeeee ee Karstsinnectes longzhouensis sp. nov. Karstsinnectes acridorsalis (Lan, 2013) Figs 1A, 2; Table 1 Oreonectes acridorsalis Lan, 2013: 68, fig. 50 (Bamu Town, Tian’e County, Hechi City, Guangxi). Troglonectes acridorsalis Xiao & Lan, 2023: 33 (Bamu Town, Tian’e County, Hechi City, Guangxi). Karstsinnectes acridorsalis Luo et al., 2023, 696 (Bamu Town, Tian’e County, Guangxi). Material examined. Paratypes. 2 ex. China; Guangx1, Hechi City, Tian’e County, Bamu Town, 22.8754°N, 107.1947°E, 284 ma.s.1. CLJH 1202001, CLJH 04100607, 37.9-38.1 mm SL, deposited in the Fishery and Animal Husbandry Bureau of Du’an, Guangxi, China. Diagnosis. Karstsinnectes acridorsalis differs from K. anophthalmus by caudal fin forked (vs. truncated), 14 branched caudal-fin rays (vs. 12), longer and lower head (length 29.9%-32.7% of SL vs. 22.7%-24.6%, height 34.6%-39.6% of head length vs. 41.0%—44.0%), longer pectoral fin (61.6%-67.7% of distance between pecto- ral-fin origin and pelvic-fin origin vs. 35.9%—48.9%); from K. hyalinus by scaleless (vs. scaled), five branched anal-fin rays (vs. four), 14 branched caudal-fin rays (vs. 11 or 12); from K. parvus and K. longzhouensis sp. nov. by lateral line absent (vs. present), lower body (body depth 13.9%-15.5% of SL vs. 17.1%-18.4% and 16.1%-— 19.4%, respectively), shorter caudal peduncle (length 15.7%-15.9% of SL vs. 18.4%-22.3% and 17.3%- 19.5%, respectively). Description. Body elongated, head depressed, snout depressed, forehead raised, head height at nostril 46.1%-— 67.9% of maximum head height. Body trunk compressed, with maximum body depth anterior to dorsal-fin origin, deepest body depth 13.9%—-15.5% of SL. Dorsal profile of head and predorsal profile slightly convex, clearly concave from dorsal-fin origin to tip of dorsal fin, and gradually convex from tip of dorsal fin to anterior quarter of caudal fin due to caudal adipose keel on upper edge of caudal peduncle. Ventral profile of head straight, near- ly straight from pectoral-fin insertion to anal-fin origin, convex from posterior margin of anal-fin base to anterior quarter of caudal fin due to caudal adipose keel on lower edge of caudal peduncle. Anterior and posterior nostrils adjacent, distance less than posterior nostril diameter, base of anterior nostril tube-shaped and tip not elongated to barbel-like. Eyes absent. Mouth inferior, snout rounded, upper and lower lips smooth, lower lip with V-shaped median notch. Three pairs of barbels, inner and outer rostral barbels reaching mouth corner, and maxillary barbel reaching anterior margin of interopercle. Inner gill rakers on first gill arch nine (One specimen). Dorsal fin with three unbranched and eight branched rays, distal margin straight, origin posterior to pelvic-fin origin, predorsal length 54.3%—55.5% of SL. Pectoral fin with one unbranched and 10 branched rays, pectoral-fin length 61.6%—67.7% of distance between pectoral-fin or- igin and pelvic-fin origin. Pelvic fin with one unbranched and five branched rays, tip of pelvic fin not reaching anus. Anal fin with two unbranched and five branched rays, dis- tal margin straight. Anus abutting anal-fin base. Caudal fin forked. High caudal adipose keels on upper and lower edges of caudal peduncle, height at most of upper adipose keel nearly 1/2 caudal peduncle depth. Caudal peduncle depth 46.1%-51.9% of its length (containing adipose keels). Lateral line and cephalic sensory pores absent. Body scaleless. Coloration. Whole body translucent, without color pattern. Fin membrane hyaline. Distribution and habitat. Only known from the type locality. Karstsinnectes acridorsalis inhabits a subterra- nean river, 22.8754°N, 107.1947°E, 284 m a.s.l. Co-in- habitants of the stream include 7riplophysa tianeensis Chen, Cui & Yang, 2004, Sinocyclocheilus furcodorsalis Chen, Yang & Lan, 1997, and Hongshuia megalophthal- mus (Chen, Yang & Cui, 2006). A “0 28°0'0"N: , Sf & a avian A ; = d y af \5 aA ce A nh 4 tk J \ f NAL w ialios: se ma SL ) ee. K. acridorsalis ¢ , 2 ad LK. parvus MY tees ad rod MT Ky > pA fe alll K. longzhouensis a a ¥ ee Figure 1. Collection sites (A), K. anophthalmus (red round), K. acridorsalis (green triangle), K. hyalinus (purple rhombus), K. longzhouensis sp. nov. (blue square), K. parvus (black star), and habitat of Karstsinnectes longzhouensis sp. nov. (B) and K. parvus (C). zse.pensoft.net 666 a mie Ge, J.-Y. et al.: A taxonomic revision of Karstsinnectes Figure 2. Lateral, dorsal, and ventral views of Karstsinnectes acridorsalis, holotype, CLJH04100608 (photograph from J.H. Lan). Scale bar: 1 cm. Remarks. The population of K. acridorsalis is small. Initially described by Lan et al. (2013) based on three specimens, subsequent collections yielded only one spec- imen, collected in 2019 by J.J. Zhou. Karstsinnectes hyalinus (Lan, Yang & Chen, 1996) Figs 1A, 3; Table 1 Heminoemacheilus hyalinus Lan, Yang & Chen, 1996, 109-112 (Bao’an Township, Du’an County, Hechi City, Guangxi). Karstsinnectes hyalinus Luo et al., 2023: 696 (Bao’an Township, Du’an County, Hechi City, Guangxi). Material examined. 3 ex. China; Guangxi, Hechi City, Du’an County, Bao’an Township, 24.0709°N, 107.9027°E, 283 m a.s.l. GXNU 94098009-94098011, 38.9-40.2 mm SL. Diagnosis. Karstsinnectes hyalinus can be distin- guished from other members of Karstsinnectes based on body-scaled (vs. scaleless). It can be further differentiated from K. anophthalmus by caudal fin forked (vs. truncat- ed); from K. acridorsalis, Karstsinnectes longzhouensis Sp. nov., and K. parvus by seven branched dorsal-fin rays (vs. eight in K. acridorsalis and nine in Karstsinnectes longzhouensis sp. nov. and K. parvus). zse.pensoft.net Description. Body elongated, head depressed. Body trunk compressed, dorsal profile of head and predorsal profile gradually convex, with maximum body depth in middle of pectoral-fin origin and pelvic-fin origin. After dorsal-fin base, dorsal profile of caudal peduncle convex due to developed caudal adipose keel on upper edge of caudal peduncle. Ventral profile of head straight, slight- ly convex from pectoral-fin origin to pelvic-fin origin, straight from pelvic-fin origin to caudal-fin base. Anterior and posterior nostrils adjacent, distance less than posterior nostril diameter, base of anterior nostril tube-shaped and tip not clearly elongated to barbel-like. Eyes absent. Mouth inferior, snout obtuse, upper and lower lips smooth. Three pairs of barbels, inner rostral barbel reaching mouth corner, outer rostral and maxillary barbels reaching anterior margin of interopercle. One specimen with one outer and 12 inner gill rakers on first gill arch. Dorsal fin with two branched and seven branched rays, distal margin straight, origin slightly anterior to pelvic-fin origin. Pectoral fin with one unbranched and 11 branched rays, pectoral-fin length 66.1%-74.8% of distance between pectoral-fin origin and pelvic-fin or- igin. Pelvic fin with one unbranched and five branched rays, tip of pelvic fin reaching anus. Anal fin with two un- branched and four branched rays, distal margin straight. 667 Zoosyst. Evol. 100 (2) 2024, 663-673 Table 1. Morphometric and meristic data of the genus Karstsinnectes. Range, mean, and standard deviation (mean + standard de- viation (SD)). K. acridorsalis (N = 2) K. anophthalmus (N = 4) K. hyalinus (N = 3) Karstsinnectes longzhouensis sp. nov. (N = 4) K. parvus (N = 3) Total length (mm) 44.8-45.9 (45.3 + 0.8) 30.5-42.8(36.4+5.1) 47.4-47.5(47.4 0.1) 57.8-73.3 (64.6 + 6.8) 32.3-34.8 (33.4 + 1.3) Total length (mm) Standard length (mm) 37.9-38.1 (38.0 + 0.1) 30.5-42.8 (36.4 + 5.1) 25.3-36.9 (31.4 + 4.8) 47.4-47.5 (47.4 + 0.1) 38.9-40.2 (39.5 + 1.0) Percentage of standard length (%) 57.8-73.3 (64.6 + 6.8) 49.4-60.1 (53.6 + 4.8) 32.3-34.8 (33.4 + 1.3) 26,5-28,.3 (27.1 £ 1:1) Deepest body depth Lateral head length Prodorsal length Prepelvic length Preanal length Preanus length Caudal peduncle length Caudal peduncle depth (containing caudal adipose keels) Head width 13.9-15.5 (14.7 + 1.1) 2990-3257 (al 3 22,0) 54.4-55.5 (54.9 + 0.8) 52.9-54.6 (53.7 + 1.2) 74.2-75.1 (74.7 + 0.6) 67.5-70.8 (69.1 + 2.3) 15.7-15.9 (15.8 + 0.1) 7.3-8.3 (7.8 + 0.7) 11.7-12.3 (12.0 + 0.4) 11.0-14.4 (13.2 + 1.6) 22.7-24.6 (23.5 + 0.8) 59.5-62.6 (60.5 + 1.4) 57.4-58.3 (57.8 + 0.5) 68.0-76.3 (73.7 + 3.9) 13.2-17.0 (15.4 + 1.7) 5.7-8.7 (6.9 + 1.3) 14.1-16.8 (15.3 + 1.2) 19.9-23.6 (21.6 + 1.8) 27.1-29.2 (28.0 + 1.1) 55.9-59.0 (57.7 + 1.6) 52.2-56.8 (55.1 + 2.6) 74.4-79.8 (77.8 + 2.9) 70.6-74.0 (72.5 + 1.7) 15.1-16.2 (15.7 + 0.5) 13.0-14.7 (13.8 + 0.9) 16.1-17.6 (16.7 + 0.8) 16.1-16.4 (17.8 + 1.4) 30.2-34.0 (32.2 + 1.6) 99.1-57.3:(96.2e2-1.1) 55.6-58.3 (57.4 + 1.3) 74.7-76.0 (75.4 + 0.6) 69.4—71.8 (70.2 + 1.1) 17,3-19.5:(18.1. + 1.0) 9.21 AOS ee Ty) 17.7-21.5 (19.4 + 1.6) 17.1-18.4 (17.9 + 0.7) 32.5-33.0 (32.7 + 0.2) 52.9-56.3 (54.8 + 1.8) 53.5-58.1 (56.0 + 2.3) 71.8-76.1 (74.6 + 2.4) 61.7-74.1 (69.2 + 6.6) 18.4-22.3 (19.9 + 2.1) 8.49.8 (9.2 + 0.7) 14.9-18.0 (16.8 + 1.7) Percentage of lateral head length (%) Head depth Head width Caudal peduncle depth (containing caudal adipose keels) Pectoral-fin length Dorsalkfin rays Pectoral-fin rays Pelvic-fin rays Anal-fin rays 34.6-39.6 (37.1 + 3.5) 37.5-39.2 (38.4 + 1.2) 46.1-51.9 (49.0 + 4.1) 41.0-44.0 (42.6 + 1.3) 60.0-73.4 (65.5 + 6.0) 45.1-48.5 (46.9 + 1.7) 59.5-60.3 (59.8 + 0.4) Percentage of caudal-peduncle length (%) 33.6-57.2 (45.4 + 9.9) 81.3-97.4 (87.8 + 8.5) 42.3-49.1 (45.4 + 3.0) 52.1-71.1 (60.4 + 8.2) 47.0-66.1 (58.5 + 8.1) Percentage of distance from pectoral-fin origin to pelvic-fin origin 61.6-67.7 (64.6 + 4.3) 3, 8-9 164.0 12 Sve 14 35.9-48.9 (40.5 + 6.1) Det 1,10 1,4 35D 12 66.1-74.8 (70.8 + 4.4) 3,7 72.4-85.9 (77.9 + 5.8) 3,9 1, 11-12 1. 3,5 13-14 40.8-44.6 (42.7 + 1.9) 45.8-54.5 (51.3 + 4.8) 37.7-53.1 (46.8 + 8.1) 67.5-76.8 (73.3 + 5.0) 3,9 1, 10 1,6 3}. 9 Caudalfin branched rays - 12-13 Figure 3. Lateral, dorsal, and ventral views of K. hyalinus, holotype KIZ1994000011 (photograph from R. Min). Scale bar: 1 cm. zse.pensoft.net 668 Anus abutting anal-fin base. Caudal fin forked, with 11 or 12 branched rays. High caudal adipose keels on upper and lower edges of caudal peduncle, height at most of upper adipose keel nearly 1/2 caudal peduncle depth. Caudal peduncle length 102.7%—123.1% of its depth (containing adipose keels). Lateral line and cephalic sensory pores absent. Body covered by scales, except head and thorax. Coloration. Whole body translucent, without color pattern. Fin membrane hyaline. Distribution and habitat. Known only from the type locality, China; Guangxi, Hechi City, Du’an County, Bao’an, 24.0709°N, 107.9027°E, 283 m as.l. The cave water in which this species resides serves as the only source of drinking water for nearby residents. The de- cline in its population is primarily attributed to habitat alterations resulting from the extraction of cave water for domestic purposes (Zhang and Cao 2021). Remarks. The population of K. hyalinus is extreme- ly small, with its presence currently known only through type specimens collected in 1994, with no additional specimens gathered since. Although the lateral line of K. hyalinus was mentioned in the original description, a re-examination of all type specimens by R. Min, the ad- ministrator of the Fish Collection Room, Kunming Nat- ural History Museum of Zoology, Kunming Institute of Zoology, revealed no obvious lateral line pores. Hence, K. hyalinus is described as lacking a lateral line 1n this study. Karstsinnectes longzhouensis Ge, Du & Zhou, sp. nov. https://zoobank.org/7D4659FE-C737-4F36-A 56F-7DB474246352 Figs 1A, B, 4; Table 1 Type materials. Holotype. China (permanent whole spec- imen in 75% alcohol); Guangxi, Chongzuo City, Long- zhou County, Xiadong Town; 22.4222°N, 106.6385°E, 170 ma.s.l.; collected by Z.Q. Nong and J.J. Zhou, 29 De- cember 2022. Kunming Natural History Museum of Zo- ology, KIZ 2023000001, 50.6 mm standard length (SL). Paratypes. China (permanent whole specimens in 75% alcohol); same collection data as for holotype, collected by Z.Q. Nong and J.J. Zhou, 29 December 2022: KIZ 2023000002-04, 3 ex., 49.4-60.1 mm SL. Diagnosis. Karstsinnectes longzhouensis sp. nov. can be distinguished from all other members of Karstsinnect- es based on the combination characteristics of the lateral line present, 11 or 12 branched pectoral-fin rays, and five branched pelvic-fin rays. It can be further distinguished from K. anophthalmus by caudal fin forked (vs. truncated), five branched pelvic-fin rays (vs. four), nine branched dorsal-fin rays (vs. seven), 13 or 14 branched caudal-fin rays (vs. 12); from K. acridorsalis by nine branched dorsal-fin rays (vs. eight), body depth 16.1%-19.4% of SL (vs. 13.9%-15.5%),; from K. hyalinus by body scaleless (vs. scaled), lateral line present (vs. absent), nine branched dorsal-fin rays (vs. sev- en), five branched anal-fin rays (vs. four); from K. parvus by 11 or 12 branched pectoral-fin rays (vs. 10), five branched pelvic-fin rays (vs. six), and uncorrected p distance is 3.9%. zse.pensoft.net Ge, J.-Y. et al.: A taxonomic revision of Karstsinnectes Description. Morphometric data of the type speci- mens of Karstsinnectes longzhouensis sp. nov. are giv- en in Table 1. Body elongated, head depressed, forehead raised, head height at nostril 60.8%—71.0% of maximum head height. Body trunk compressed, with maximum body depth in middle of pectoral-fin origin and pelvic-fin origin, deepest body depth 16.1%-19.4% of SL. Dorsal profile of forehead and predorsal profile convex, concave from dorsal-fin origin to anterior margin of upper caudal adipose keel. Caudal adipose keel on upper edge of cau- dal of peduncle slightly convex. Ventral profile of head straight, slightly convex from pectoral-fin origin to pel- vic-fin origin, straight between pelvic-fin and anal-fin or- igin, and gradually concave from anal-fin base to anterior quarter of caudal fin due to caudal adipose keel on lower edge of caudal peduncle. Anterior and posterior nostrils adjacent, distance less than posterior nostril diameter, base of anterior nostril tube-shaped and tip not elongated to barbel-like. Eyes absent. Mouth inferior, snout rounded, upper and lower lips smooth, lower lip with V-shaped median notch. Three pairs of barbels, inner rostral barbel reaching anterior nostril, outer rostral barbel reaching posterior margin of posterior nostril, and maxillary barbel reaching anterior margin of interopercle. Two specimens with 11—12 inner gill rakers on first gill arch. Dorsal fin with three unbranched and nine branched rays, distal margin of dorsal fin straight, origin anterior to pelvic-fin origin, predorsal length 55.1%-57.3% of SL. Pectoral fin with one unbranched and 10 branched rays, pectoral-fin length 72.4%-—85.9% of distance be- tween pectoral-fin origin and pelvic-fin origin. One un- branched and five branched pelvic-fin rays, tip of pel- vic fin reaching, but not exceeding anus. Anus abutting anal-fin base. Caudal fin forked, with 13 or 14 branched caudal-fin rays. High caudal adipose keels on upper and lower edges of caudal peduncle, height at most of upper adipose keel less than 1/2 caudal peduncle depth. Caudal peduncle length 151.2%—212.9% of its depth (containing adipose keels). Lateral line and head sensory pores ab- sent. Body scaleless. Coloration. Dorsal and trunk of body yellowish, ab- domen gray and translucent, stomach and intestine visible from outside. Without color pattern. Fin membrane hyaline. Distribution and habitat. Karstsinnectes longzhouen- sis sp. nov. inhabits karst caves located in the Guangx1 Qinglongshan provincial natural reserve, specifically in Xiadong Town, Longzhou County, Chongzuo City, Guangxi, China (22.4222°N, 106.6385°E, 171 m as.l.). The species was observed in a subterranean pool accessed through an oval cave entrance and a narrow passage. The pool water depth exceeded 1 m and was characterized by a substratum of mud and cobblestones. Etymology. The specific name “/ongzhouensis” is de- rived from the Chinese name of the type locality in Long- zhou County. Therefore, the Chinese and English com- mon names for this new species are “J& | -H 42M fil” and “Longzhou Chinese Karst Loach,” respectively. Zoosyst. Evol. 100 (2) 2024, 663-673 669 Figure 4. Lateral, dorsal, and ventral views of Karstsinnectes longzhouensis sp. nov., holotype KIZ2023000001. Scale bar: 1 cm. Remarks. On 29 December 2022, Z.Q. Nong collect- ed type specimens within a karst cave. By May 2023, the cave showed significant signs of drying. In another cave located 300 m away, J.J. Zhou collected a deteriorated specimen. Decreased precipitation and the removal of domestic water from the cave appear to have negatively influenced the viability of cavefish during the dry season. Karstsinnectes parvus (Zhu & Zhu, 2014) Figs 1A, C, 5; Table 1 Heminoemacheilus parva Zhu & Zhu, 2014: 18-21 (Ande Town, Napo County, Guangx1). Karstsinnectes parvus Luo et al., 2023, 696 (Ande Town, Napo County, Guangxi). Neotype designation. Both holotype and paratypes were originally deposited at the Guangxi Fisheries and Animal Husbandry School under registration numbers 2011006— 2011009 (Zhu and Zhu 2014) but were broken and lost three years ago (Y. Zhu, pers. comm.). Conforming with Article 75.3 of the Code ICZN 1999), a neotype from the type locality is herein designated (Fig. 5). Neotype. China; Guangxi, Baise City, Napo County, Nongma Village, 23.1803°N, 106.0020°E, 934 m a.s.l., collected by J.J. Zhou, J.Q. Luo, X.M. Luo, and Z.X. Qin on 1 May 2023; KIZ 2023000005 (Fig. 5), 26.5 mm SL. Non-type material. 2 ex. China; same collected with neotype, collected by J.J. Zhou, J.Q. Luo, X.M. Luo, and Z.X. Qin on 1 May 2023; GXNU 20230501001, GXNU 20230501003, 26.5—28.3 mm SL. Diagnosis. Karstsinnectes parvus can be distinguished from K. acridorsalis by lateral line present (vs. absent), nine branched dorsal-fin rays (vs. eight), six branched pelvic-fin rays (vs. five), 12 or 13 branched caudal-fin rays (vs. 14); from K. anophthalmus by caudal fin forked (vs. truncated), lateral line present (vs. absent), nine branched dorsal-fin rays (vs. seven), six branched pel- vic-fin rays (vs. four); from K. hyalinus by body scale- less (vs. scaled), lateral line present (vs. absent), nine branched dorsal-fin rays (vs. seven), five branched anal- fin rays (vs. four); from Karstsinnectes longzhouensis sp. nov. by 10 branched pectoral-fin rays (vs. 11 or 12), six branched pelvic-fin rays (vs. five). Description. Body elongated, slightly flattened in front, strongly compressed in back. Maximum body depth anterior to dorsal-fin origin, deepest body depth 17.1%-18.4% of SL. Head depressed and flattened, maximum width greater than maximum depth. Anterior and posterior nostrils adjacent, distance less than poste- rior nostril diameter, base of anterior nostril tube-shaped and tip not elongated to barbel-like. Eyes absent. Mouth inferior, snout rounded, upper and lower lips smooth, lower lip with V-shaped median notch. Three pairs of barbels, inner rostral barbel reaching anterior nostril, outer rostral barbel reaching posterior margin of posteri- or nostril, and maxillary barbel reaching anterior margin of interopercle. One specimen with 11 inner gill rakers on first gill arch. Dorsal fin with three unbranched and nine branched rays, distal margin of dorsal fin straight, origin anterior to pelvic-fin origin, predorsal length 52.9%-56.3% of SL. Pectoral fin with one unbranched and 10 branched zse.pensoft.net 670 Ge, J.-Y. et al.: A taxonomic revision of Karstsinnectes Figure 5. Lateral, dorsal, ventral views, and living photo of K. parvus, neotype KIZ2023000005 (photograph from M. Liang). Scale bar: 1 mm. rays, pectoral-fin length 67.5%-76.8% of distance between pectoral-fin origin and _ pelvic-fin origin. Pelvic fin with one unbranched and six branched rays, tip of pelvic fin exceeding anus. Anal fin with three un- branched and five branched rays, distal margin straight. Anus abutting anal-fin base. Caudal fin forked, with 12 or 13 branched rays. High caudal adipose keels on upper and lower edges of caudal peduncle, height at most of upper adipose keel less than 1/2 caudal peduncle depth. Caudal peduncle length 188.4%—265.6% of its depth (containing adipose keels). Lateral line present. Body scaleless. Coloration. Dorsal and trunk of body gray and trans- lucent, stomach and intestine visible from outside. With- out color pattern. Fin membrane hyaline. Distribution and habitat. Karstsinnectes parvus in- habits a karst cave in Nongma Village, Napo County, Baise City, Guangxi, China; 23.1803°N, 106.0020°E, 934 m a.s.l., in a small and shallow river (approximately 300 m long, depths of less than 20 cm), characterized by substrata composed of mud and cobblestones. Five to six specimens were caught in each survey in 2021. zse.pensoft.net Remarks. Given the loss of the type specimens three years ago (Y. Zhu, pers. comm.), three specimens of K. parvus were newly collected from the type locality. These specimens conformed to the original description in all aspects except for the caudal fin count. The caudal fin of the holotype was damaged in the original account, pre- venting verification of the fin ray count from the holotype photograph in the initial description. Lan et al. collected this species from the type locality in 2021 and noted 13 branched rays of the caudal fin (Xiao and Lan 2023). This observation suggests that the unbranched rays of the cau- dal fin may have been included in the count of branched rays in the original description. Genetic comparisons Based on BI analyses, molecular phylogenies demon- strated that species of Karstsinnectes constituted a monophyletic group with robust support (100% boot- straps). Furthermore, they were sister to the clade com- Zoosyst. Evol. 100 (2) 2024, 663-673 400 Leptobotia elongata JQ230103 Parabotia fasciata KM393223 671 ia. = Petruichthys brevis NCO31637 Schistura reticulofasciata NC034745 100) dee Schistura sikmaiensis NC034746 Homatula potanini KP749475, Barbatula nuda KF574248 Po CT a Triplophysa xiangxiensis NCO29492 100 100) a Traccatichthys pulcher KF 765806 Lefua costata NC029385 Lefua nikkonis NCO27662 6S 00 or Eonemachilus longidorsalis OMT32331 400) Eonemachilus niger NCO63106 100 100) 100} 4100) Troglonectes furcocaudalis NCO32384 100 Troglonectes dagikongensis MG738689 Troglonectes longibarbatus MT631977 Paranemachilus Jinxiensis NC039380 Paranemachilus pingguoensis MWO043720 Paranemachilus zhengbaoshani ON116530 aranemachilus chongzuo MW532082 Karstsinnectes acridorsalis ON116515 100;— Karstsinnectes anophthalmus ON116513 __ Karstsinnectes anophthalmus ON116506 400 Karstsinnectes longzhouensis sp. nov. OR947935 Karstsinnectes parvus ON116520 Oreonecies platycephatus NCO31579 Oreonectes polystignus ON116514 Oreonectes guananensis ON116507 Oreonectes luochengensis ON116495 Micronemacheilus cruciatus NCO33960 4007-— Micronemacheilus pulcherrimus 0Q024374 Micronemacheilus puicherrimus MK387705 0.03 400) Guinemachilus sp. NCO61031 4 Guinemachilus fongibarbatus ON116508 100 Guinemachilus longibarbatus ON148334 100, Guinemachilus pseudopulcherrimus OQ024377 : Ir Guinemachilus pseudopulcherrimus OQ024375 95 Guinemachilus pseudopulcherrimus 0Q024376 Figure 6. Bayesian phylogram of Karstsinnectes based on the mitochondrial genomes of 40 nemacheilid and two botiid species (outgroups). The numbers above the branches are Bayesian posterior probabilities (BPP%). prised of Oreonectes, Micronemacheilus, and Guine- machilus species. Karstsinnectes longzhouensis sp. nov. was determined to be a sister group to K. parvus and further sister to K. anophthalmus and K. acridorsalis (Fig. 6). Additionally, pairwise comparisons of com- plete mitochondrial genomes revealed that the average uncorrected p distance between species of Karstsin- nectes ranged from 3.96% to 11.38% (average 9.65%). The minimum uncorrected p distance is between K. longzhouensis sp. nov. and K. parvus (3.96%), and the maximum uncorrected p distance is both between K. acridorsalis and K. anophthalmus (11.38%) and be- tween K. acridorsalis and K. parvus (11.38%) (Table 3). In consideration of both molecular and morphological comparisons, we confidently assign the new species to the genus Karstsinnectes. Principal component analysis (PCA) The first two principal components (PCs) explained 71.1% of the variance (Table 2). The first principal com- ponent (PC1) accounted for 46.7% of the morphological variation and distinguished variables such as body depth/ SL, head lateral length/SL, caudal peduncle depth/SL, pectoral-fin length/SL, and pelvic-fin length/SL. Addi- tionally, it separated pectoral-fin length relative to the distance between pectoral- and pelvic-fin origins and pel- vic-fin length relative to the distance between pelvic- and anal-fin origins. Predorsal length/SL demonstrated a pos- itive correlation with PC1 scores, while predorsal length/ SL exhibited a negative correlation, with factor loadings exceeding 0.60. The second factor (PC2) accounted for 24.4% of the morphological variation, distinguishing variables such as head width to SL, head depth to head lateral length, and head width to head lateral length, which all showed a positive correlation with PC2 scores. Conversely, caudal peduncle length to caudal peduncle depth was negatively correlated with PC2 scores. Scat- ter plot analysis revealed that species within the genus Karstsinnectes could be differentiated based on their morphometric traits (Fig. 7). Table 2. Loadings of the first three PCs for the morphometric characters of Karstsinnectes. * loadings > 60%. Character PC1 PC2 PC3 Body depth/SL 0.871* 0.271 -0.126 Head lateral length/SL 0.798* -0.445 0.221 Prodorsal length/SL -0.704* 0.592 0.099 Propelvic length/SL 0.314 0.387 0.577 Preanal/SL 0.392 0.343 = -0.310 GPISle 0.489 0.415 0.690* CPD/SL 0.830* 0.455 -0.242 Head width/SL 0.462 0.662* 0.498 Pectoral-fin length/SL 0.912* 0.157 0.146 Pelvic-fin length/SL 0.883* -0.195 -0.247 Head depth/head lateral length 0.404 0.779* 0.248 Head width/head lateral length 0.206 0.896* 0.257 Pectoral-fin length/distance between 0.912* -0.193 0.083 pectoralfn and pelvic-fin origin Pelvic-fin length/distance between pelvic-fin 0.858* -0.274 0.075 and anakfin origin CPL/CPD 0.579 -0.607* 0.522 Prp. Tot1 46.70% 24.40% 11.90% Table 3. Uncorrected pairwise distances between species of Karstsinnectes based on complete mitochondrial genomes. a 2 3 1K. anophthalmus 2K. acridorsalis 0.1138 3K. longzhouensis 0.1008 0.1071 4K. parvus 0.1036 0.1138 0.0396 zse.pensoft.net 672 200000 & K, Jongzhouensis @® K. parvus ¥ K. acnoorsalis * K. anophthaimus © K. hyalinus = 1.00000 + e a os + mH 0 nonod t + ry * 1 2G0o0 - : 00000 ¥ -nponea 150000 -1 00000 - 50000 oO000 =0000 1.00000 Figure 7. Scatter plots of first and second PCs of pooled mor- phometric data of Karstsinnectes. Discussion While Luo et al. (2023) established the genus Karstsin- nectes, its diagnosis remains unclear. Du et al. (2023) indicated that the location of the anterior and posteri- or nostrils serves as an important characteristic for the generic diagnosis of Chinese nemacheilids with tube- shaped anterior nostrils and can be categorized into three types based on their location, namely separated, adjacent, and closed-set (Du et al. 2023). The anterior nostrils of Karstsinnectes are tube-shaped and located adjacent to the posterior nostrils, with the intervening distance shorter than the diameter of the posterior nostril. Addi- tionally, the tip of the anterior nostril does not extend in a barbel-like manner. Therefore, the primary diagnostic features of Karstsinnectes include anterior nostril tube- shaped, adjacent to posterior nostril, anterior nostril tip not elongated to barbel-like, lips with furrows, caudal pe- duncle with adipose crests, and bony capsule of the swim bladder open posteriorly. In morphology, K. anophthalmus possesses distinct characteristics from other congeneric species, including an obtuse snout, shorter pectoral and pelvic fins, a pec- toral-fin length less than half the distance from its origin to the pelvic-fin origin, and a pelvic fin tip that does not reach the anus. Scatter plots of the first and second PCs of pooled morphometric data of Karstsinnectes also indicat- ed the morphological difference of K. anophthalmus. The morphological difference could indicate that K. anoph- thalmus represents a new genus. However, the molecular analysis 1s inconsistent with the morphological evidence. In the phylogenetic tree, K. Jongzhouensis sp. nov. was determined to be a sister group to K. parvus and further sister to K. anophthalmus and K. acridorsalis. According to Luo et al. (2023), the specimens collected from Daxin County, Chongzuo City, Guangxi, China, were identified as K. anophthalmus. However, J.H. Lan and J. Yang ob- served that the morphology of the specimens from Daxin differed from those found in the type locality. However, the samples of ON116513 and ON116506 were from Daixin zse.pensoft.net Ge, J.-Y. et al.: A taxonomic revision of Karstsinnectes County; the specimens were not sequenced from the type locality. Therefore, further investigations are required to determine the taxonomic status of these specimens. Blind cavefish represent a powerful natural model for studying adaptations in extreme environments. Cave hab- itats, characterized by limited food resources, low oxygen levels, and perpetual darkness (Zhao and Zhang 2009), present considerable challenges. To adapt and thrive in such environments, cavefish have undergone a series of evolutionary changes, including eye and pigmentation loss, enhancement of non-visual senses, development of specialized jaws for feeding, increased number of taste buds and barbels, and enhanced ability to store adipose tissue (Yang et al. 2016; Ma et al. 2019, 20233). Blind cavefish within the genus Karstsinnectes exhibit a suite of troglomorphic traits, including scaleless and colorless bod- ies, absence of eyes, elongated snouts (except K. anoph- thalmus), and caudal peduncles with well developed adi- pose crests. Current studies suggest that surface-dwelling species, upon colonizing cave environments and being granted sufficient time, undergo analogous transforma- tions. Consequently, cave-dwelling organisms serve as a valuable natural framework for investigating convergent and adaptive evolutionary processes. The survival of cavefish is significantly threatened by human activities, climate change, water pollution, and invasive species. As of 2021, the Red List of Bio- diversity in China includes 13 cavefish species from the Nemacheilidae family (Zhang and Cao 2021). Zhang and Cao (2021) stated that the population of K. anophthalmus is very small, with only one or two specimens caught in each survey in 1996, 2003, and 2010, respectively. Major threats include road infrastructure construction, tourism activities, and groundwater contamination from agricul- tural operations in the recharge areas of the cave (Zhang and Cao 2021). Additionally, Shu et al. (2013) highlight- ed the impact of droughts and human extraction of wa- ter from cave pools on cave biodiversity in southwestern China. Thus, to conserve these valuable cavefish species, the implementation of conservation measures, such as habitat protection, artificial breeding, and fundamental research, 1s crucial. Conclusions In this paper, a new blind species is described from Guangxi, China. The phylogenetic tree indicated that the new species belongs to the genus Karstsinnectes. Addi- tionally, the genus diagnosis is redefined, a neotype for K. parvus is designated due to the type specimens lost, and the morphology character of K. hyalinus 1s revised. The population of these species is quite small and sensi- tive to human activity. The protection of cave fish is often neglected due to the difficulty of cave exploration. The findings of this study improve our understanding of the species diversity of the genus Karstsinnectes and provide the basis for cavefish protection. Zoosyst. Evol. 100 (2) 2024, 663-673 Competing interests The authors declare that they have no competing interests. Authors’ contributions J.Y.G. and L.N.D. measured the specimens, analyzed the data, and prepared the manuscript. L.N.D. and J.Y. con- ceived and designed the study, analyzed the molecular data, constructed the phylogenetic tree, and provided fund- ing for complete mitochondrial genomes and field surveys. Z.Q.N. and J.J.Z. conducted the field survey. All authors read and approved the final version of the manuscript. Acknowledgments This study was funded by the Guangxi Natural Science Foundation Project (2022GXNSFAA035563), Key Lab- oratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, China (ERESEP2022Z05), Survey and Assessment of Priority Areas for Terrestrial Biodiver- sity Conservation in Guangxi (2022-2023), and Guangxi Zhuang Autonomous Region Project of Undergraduate on Innovation and Entrepreneurship (S202310602180). We are grateful to J.H. Lan for providing photographs of K. acridorsalis, R. Min for providing photographs of K. anophthalmus and K. hyalinus, and M. Liang for tak- ing photographs of K. parvus. We thank H. Zhou, B.M. Wang, X.M. Luo, J.Q. Luo, and Z.X. Qin for collecting specimens of Karstsinnectes. References Beier S, Thiel T, Miinch T, Scholz U, Mascher M (2017) MISA-web: A web server for microsatellite prediction. 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