Research Article
Print
Research Article
Two new hypogean species of the genus Triplophysa (Osteichthyes, Cypriniformes, Nemacheilidae) from Guizhou Province, Southwest China, with underestimated diversity
expand article infoChang-Ting Lan‡, Li Wu‡, Tao Luo‡, Ye-Wei Liu§, Jia-Jun Zhou|, Jing Yu‡, Xin-Rui Zhao‡, Ning Xiao#, Jiang Zhou‡
‡ Guizhou Normal University, Guiyang, China
§ Guangxi University, Nanning, China
| Zhejiang Forest Resource Monitoring Center, Hangzhou, China
¶ Zhejiang Forestry Survey Planning and Design Company Limited, Hangzhou, China
# Guiyang Healthcare Vocational University, Guiyang, China

Corresponding author: Tao Luo ( 18786413778@163.com )

Academic editor: Nina Bogutskaya
© 2024 Chang-Ting Lan, Li Wu, Tao Luo, Ye-Wei Liu, Jia-Jun Zhou, Jing Yu, Xin-Rui Zhao, Ning Xiao, Jiang Zhou.
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.
Citation: Lan C-T, Wu L, Luo T, Liu Y-W, Zhou J-J, Yu J, Zhao X-R, Xiao N, Zhou J (2024) Two new hypogean species of the genus Triplophysa (Osteichthyes, Cypriniformes, Nemacheilidae) from Guizhou Province, Southwest China, with underestimated diversity. ZooKeys 1214: 237-264. https://doi.org/10.3897/zookeys.1214.122439
ZooBank: urn:lsid:zoobank.org:pub:8B42B493-BD13-4D4C-9161-55978636D055
Open Access

Abstract

Two new species of the genus Triplophysa from southwestern Guizhou Province, China, are described. These are Triplophysa ziyunensis Wu, Luo, Xiao & Zhou, sp. nov. and Triplophysa yaluwang Lan, Liu, Zhou & Zhou, sp. nov. from Maoying Town, Ziyun County, Guizhou Province, China. Triplophysa ziyunensis Wu, Luo, Xiao & Zhou, sp. nov. is distinguished from other hypogean species of the genus Triplophysa by having a combination of the following characteristics: body naked, scaleless, pigmented markings on surface of body, except ventral; eyes reduced, diameter 2.4–4.9% of head length; pelvic-fin tip extending to anus; tip of pectoral fin not reaching pelvic fin origin; anterior and posterior nostrils closely set, with anterior nostril elongated to a barbel-like tip; tip of outrostral barbel extending backward, not reaching anterior margin of the eye; lateral line complete; posterior chamber of air bladder degenerated; and fin differences. Triplophysa yaluwang Lan, Liu, Zhou & Zhou, sp. nov. is distinguished from other hypogean species of the genus Triplophysa by having a combination of the following characteristics: body naked, scaleless, with irregular pale and dark brownish brown markings, except ventrally; eyes reduced, diameter 4.6–6.1% of head length; pelvic-fin tip reaching anus; tip of pectoral fin not reaching to pelvic fin origin; anterior and posterior nostrils closely set, with anterior nostril elongated to a barbel-like tip; tip of outrostral barbel extending backward, not reaching to anterior margin of the eye; lateral line complete; posterior chamber of air bladder degenerated; and fin differences. Mitochondrial Cyt b revealed relatively small genetic differences, 1.4–2.0%, between the two new species and close relatives. Nuclear gene RAG1 indicated that the two new species possessed unique haplotypes with multiple linking mutations. This study emphasizes the importance of utilizing nuclear genes to identify new species in rapidly speciation cave species, with small genetic differences due to mitochondrial introgression occurring interspecies.

Key words

Mitochondrial DNA, morphology, new species, nuclear gene, Triplophysa

Introduction

The high-plateau loach fish genus Triplophysa Rendahl, 1933 comprises more than 184 recognized species of small loaches that are distributed on the Qinghai-Xizang Plateau and nearby regions (Luo et al. 2023; Fricke et al. 2024). Morphological characteristics that distinguish Triplophysa from other genera in the Nemacheilidae include closely set anterior and posterior nostrils and a posterior wall of the bony capsule of the swim bladder. Males have tubercle-bearing, elevated skin on both sides of the head and a thickened tuberculated pad or agglomerations on the dorsal surfaces of the broadened and widened pectoral-fin rays (Zhu 1989; Zheng et al. 2009; Prokofiev 2010; He et al. 2012; Ren et al. 2012; Yang et al. 2012; Wu et al. 2018a; Chen and Peng 2019; Deng et al. 2022). Triplophysa species are distributed from the Qinghai-Xizang Plateau at an average elevation of 4000 m to the Yunnan-Guizhou Plateau at an average elevation of 1000–2000 m (Zhu 1989; Luo et al. 2023). Their habitats include lakes, rivers, and caves (Zhu 1989; Lan et al. 2013), on the basis of which Triplophysa can be divided into two life groups: An epigean group and a hypogean or cave-dwelling group (Liu et al. 2022; Lu et al. 2022). The hypogean group is mainly distributed in underground rivers in southwest China, including Guizhou, Chongqing, Guangxi, Yunnan, and Hunan provinces (Luo et al. 2023). This group can be further subdivided into two morphological types, the stygobionts, and stygophiles (Zhao et al. 2011; Ma et al. 2019), based on the level of adaptation to the cave environment (Zhao et al. 2011; Lu et al. 2022). There are 106 species of Triplophysa and 39 hypogean species are distributed in Chongqing, Guangxi, Guizhou, Yunnan, and Hunan provinces in China (Table 1) (Lan et al. 2013; Zhang et al. 2020; Chen et al. 2021; Liu et al. 2022; Lu et al. 2022; Luo et al. 2023).

Table 1.
Download as
CSV
XLSX

A list of 39 species of hypogean fishes of the genus Triplophysa distributed in the Southwest China.

ID Species Province Main drainage Tributary Reference
1 T. aluensis Li & Zhu, 2000 Yunnan Pearl River Nanpanjiang River Li and Zhu 2000
2 T. anshuiensis Wu, Wei, Lan & Du, 2018 Guangxi Pearl River Hongshui River Wu et al. 2018a
3 T. anlongensis Lan, Song, Luo, Zhao, Xiao & Zhou, 2023 Guizhou Pearl River Nanpanjiang River Luo et al. 2023
4 T. baotianensis Li, Liu & Li, 2018 Guizhou Pearl River Nanpanjiang River Li et al. 2018
5 T. cehengensis Luo, Mao, Zhao, Xiao & Zhou, 2023 Guizhou Pearl River Beipanjiang River Luo et al. 2023
6 T. erythraea Liu & Huang, 2019 Hunan Yangtze River Yuanjiang River Huang et al. 2019
7 T. fengshanensis Lan, 2013 Guangxi Pearl River Hongshui River Lan et al. 2013
8 T. flavicorpus Yang, Chen & Lan, 2004 Guangxi Pearl River Hongshui River Yang et al. 2004
9 T. gejiuensis (Chu & Chen, 1979) Yunnan Pearl River Nanpanjiang River Chu and Chen 1979
10 T. guizhouensis Wu, He & Yang, 2018 Guizhou Pearl River Hongshui River Wu et al. 2018b
11 T. huapingensis Zheng, Yang & Che, 2012 Guangxi Pearl River Hongshui River Zheng et al. 2012
12 T. langpingensis Yang, 2013 Guangxi Pearl River Hongshui River Lan et al. 2013
13 T. longipectoralis Zheng, Du, Chen & Yang, 2009 Guangxi Pearl River Liujiang River Zheng et al. 2009
14 T. longliensis Ren, Yang & Chen, 2012 Guizhou Pearl River Hongshui River Ren et al. 2012
15 T. luochengensis Li, Lan, Chen & Du, 2017 Guangxi Pearl River Hongshui River Li et al. 2017a
16 T. macrocephala Yang, Wu & Yang, 2012 Guangxi Pearl River Liujiang River Yang et al. 2012
17 T. nandanensisLan, Yang & Chen, 1995 Guangxi Pearl River Hongshui River Lan et al. 1995
18 T. nanpanjiangensisZhu & Cao, 1988 Yunnan Pearl River Nanpanjiang River Zhu and Cao 1988
19 T. nasobarbatula Wang & Li, 2001 Guizhou Pearl River Liujiang River Wang and Li 2001
20 T. panzhouensis Yu, Luo, Lan, Xiao & Zhou, 2023 Guizhou Pearl River Nanpanjiang River Luo et al. 2023
21 T. posterodorsalus (Li, Ran & Chen, 2006) Guangxi Pearl River Liujiang River Ran et al. 2006
22 T. qingzhenensis Liu, Zen, & Gong, 2022 Guizhou Yangtze River Wujiang River Liu et al. 2022
23 T. qini Deng, Wang & Zhang, 2022 Chongqing Yangtze River Yuanjiang River Deng et al. 2022
24 T. qiubeiensis Li & Yang, 2008 Yunnan Pearl River Nanpanjiang River Li et al. 2008
25 T. rongduensis Mao, Zhao, Yu, Xiao & Zhou,2023 Guizhou Pearl River Beipanjiang River Luo et al. 2023
26 T. rosa Chen & Yang, 2005 Chongqing Yangtze River Wujiang River Chen and Yang 2005
27 T. sanduensisChen & Peng, 2019 Guizhou Pearl River Duliujiang River Chen and Peng 2019
28 T. shilinensis Chen,Yang & Xu, 1992 Yunnan Pearl River Nanpangjiang River Chen et al. 1992
29 T. tianeensis Chen, Cui & Yang, 2004 Guangxi Pearl River Hongshui River Chen et al. 2004
30 T. tianlinensis Li, Li, Lan & Du, 2017 Yunnan Pearl River Hongshui River Li et al. 2017b
31 T. tianxingensis Yang, Li & Chen, 2016 Yunnan Pearl River Nanpangjiang River Yang et al. 2016
32 T. wudangensis Liu, Zen & Gong, 2022 Guizhou Yangtze River Wujiang River Liu et al. 2022
33 T. wulongensis Chen, Sheraliev, Shu & Peng, 2021 Chongqing Yangtze River Wujiang River Chen et al. 2021
34 T. xiangshuingensis Li, 2004 Yunnan Pearl River Nanpanjiang River Li 2004
35 T. xiangxiensis Yang, Yuan & Liao, 1986 Hunan Yangtze River Yuanjiang River Yang et al. 1986
36 T. xichouensis Liu, Pan, Yang & Chen, 2017 Yunnan Red River Red River Liu et al. 2017
37 T. xuanweiensis Lu, Li, Mao & Zhao, 2022 Yunnan Pearl River Beipanjiang River Lu et al. 2022
38 T. yunnanensis Yang, 1990 Yunnan Pearl River Nanpanjiang River Chu and Chen 1990
39 T. zhenfengensis Wang & Li, 2001 Guizhou Pearl River Beipanjiang River Wang and Li 2001

Guizhou Province is the region where the two major rivers of Asia, the Pearl River, and the Yangtze River, are separated (Fig. 1). The subtropical monsoon climate and paleogeology have shaped the karst landscapes and rich cave resources. This ecological background has enabled the formation of a diverse subterranean biota (Li et al. 2022; Wen et al. 2022). A total of 12 species occur in this region, eight of which have been described during the last ten years. These are Triplophysa anlongensis Lan, Song, Luo, Zhao, Xiao & Zhou, 2023, T. baotianensis Li, Liu & Li, 2018, T. guizhouensis Wu, He & Yang, 2018, T. cehengensis Luo, Mao, Zhao, Xiao & Zhou, 2023, T. longliensis Ren, Yang & Chen, 2012, T. nasobarbatula Wang & Li, 2001, T. panzhouensis Yu, Luo, Lan, Xiao & Zhou, 2023, T. qingzhenensis Liu, Zen & Gong, 2022, T. rongduensis Mao, Zhao, Yu, Xiao & Zhou, 2023, T. sanduensis Chen & Peng, 2019, T. wudangensis Liu, Zen & Gong, 2022, and T. zhenfengensis Wang & Li, 2001 (Wang and Li 2001; Ren et al. 2012; Li et al. 2018; Wu et al. 2018a; Chen and Peng 2019; Liu et al. 2022; Luo et al. 2023). Body pigmentation and pigmented markings were present in 11 of the 12 species, except for T. cehengensis, and the key distinguishing characteristics of these species are shown in Table 2. Four new species of Triplophysa were recently described in Guizhou (Luo et al. 2023), thus suggesting that additional undescribed species may exist in this region.

Figure 1. 

Sample collection localities and distributions of the two new species and 39 hypogean species of the genus Triplophysa in southern China. The base maps are from the Standard Map Service website (http://bzdt.ch.mnr.gov.cn/index.html).

Table 2.
Download as
CSV
XLSX

Comparison of the diagnostic features of the two new species described here with those selected for the 39 recognized hypogean species of the genus Triplophysa. Modified from Wu et al. (2018b), Liu et al. (2022), and Luo et al. (2023). “–” indicates uncertainty.

ID Species Body pigmentation Eyes Eye diameter (% HL) Interorbital width
(% HL)		 Dorsal fin distal margin Secondary sex characteristics Scales Lateral line Posterior chamber of air bladder Dorsal fin rays Analfin rays Pectoral fin rays Pelvic fin rays Caudalfin rays Tip of pelvic fin reaching anus Anterior nostril barbel-like Vertebrae
1 T. yaluwang sp. nov. Entire body Reduced 4.6–6.1 24.3–26.0 Emarginated Absent Absent Complete Degenerated iii, 7 iii, 5 i, 9 i, 5–6 14 Yes Yes 4 + 36
2 T. ziyunensis sp. nov. Entire body Reduced 2.4–4.9 22.3–26.2 Truncated Absent Absent Complete Degenerated iii, 8 iii, 5 i, 10 i, 6 16 Yes Yes 4 + 35
3 T. aluensis Absent Reduced 5.6 22.2 Truncated – Absent Complete Degenerated iii, 7 iii, 5 i, 9 i, 6 13 No Yes –
4 T. anlongensis Entire body Normal 5.1–9.3 32.1–35.6 Truncated Absent Absent Complete Degenerated iii, 8 iii, 5 i, 11 ii, 8 16 No Yes 4 + 37
5 T. anshuiensis Dorsal Absent Absent – Truncated Absent Absent Complete Developed iv, 7–8 ii, 6 i, 10 i, 6 14 Yes Yes –
6 T. baotianensis Entire body Normal 14.0–15.0 3.40–4.57 Truncated – Absent Complete Degenerated iii, 6–7 ii, 4–5 i, 9 i, 5 11–13 No Yes –
7 T. cehengensis Absent Reduced 1.5–2.2 27.2–36.5 Emarginated Absent Absent Complete Developed iv, 9 iii, 5 i, 10 ii, 8 16 Yes Yes 4 + 35
8 T. erythraea Absent Absent Absent – Truncated Absent Absent Complete Developed ii, 8 i, 6 ii, 10 ii, 5 17 Yes No –
9 T. fengshanensis Absent Absent Absent – Truncated – Absent Complete – ii, 8 ii, 6 i, 8–10 i, 6–7 16 No Yes –
10 T. flavicorpus Entire body Normal 5.1–6.8 3.1–5.2 Emarginated – Present Complete Degenerated iii, 10 iii, 6–7 i, 11 i, 6–7 16 Yes No 4 + 34
11 T. gejiuensis Absent Absent Absent – Truncated – Absent Complete Developed iii, 7–8 iii, 4–6 i, 10 i, 5 14–15 Yes Yes –
12 T. guizhouensis Entire body Normal 9.4–12.1 20.3–24.3 Truncated Absent Present Complete Developed iii, 8 iii, 6 i, 8–9 i, 6 14 No Yes –
13 T. huapingensis Entire body Normal 10.4–14.3 27.6–30.8 Truncated Present Present Complete Degenerated iii, 8–9 iii, 5 i, 9–10 i, 5–6 16 No No –
14 T. langpingensis Absent Reduced 2.7–5.9 30.6–34.5 Truncated – Absent Incomplete – iii, 7–8 iii, 5–6 i, 10–11 i, 6 14 Yes Yes –
15 T. longipectoralis Entire body Normal 11.8–16.4 21.2–25.3 Emarginated Present Present Complete Degenerated iii, 8 iii, 5–6 i, 9–10 i, 6 14–15 Yes Yes 4 + 35
16 T. longliensis Entire body Normal 9.5–11.5 31.4–37.5 Emarginated Present Absent Complete Developed iii, 8 iii, 5 i, 10 i, 6 15–16 Yes Yes 4 + 38
17 T. luochengensis Entire body Reduced 7.5–8.6 18.4–21.3 Truncated Present Present Complete Degenerated iii, 8 ii, 6 i, 10 i, 6 16–17 No Yes 4 + 33–34
18 T. macrocephala Entire body Reduced 3.6–8.0 22.9–25.8 Truncated Present Absent Complete Degenerated iii, 7–9 iii, 5–6 i, 9–11 i, 6 15–17 Yes Yes –
19 T. nandanensis Entire body Normal 11.1–21.3 24.4–27.8 Emarginated – Present Complete Degenerated iv, 8 iv, 5 i, 9–10 i, 6 14–16 No Yes 4 + 36
20 T. nanpanjiangensis Entire body Normal 12.0–16.5 30.3–34.5 Truncated Present Absent Complete Degenerated iii, 7–8 ii, 5 i, 9–10 i, 6 16 No Yes 4 + 38
21 T. nasobarbatula Entire body Normal 9.1–13.3 27.0–33.3 Truncated – Present Complete Degenerated iii, 8 iii, 5 i, 9 i, 6 15 Yes Yes 4 + 36
22 T. panzhouensis Entire body Normal 7.0–11.0 22.1–31.3 Truncated Absent Absent Complete Degenerated iv, 7–8 iii, 5 i, 11 ii, 7 16 No Yes 4 + 35
23 T. posterodorsalus Absent Absent Absent – Truncated – Absent Complete – iii, 6 ii, 4 i, 13 i, 5 15 No Yes –
24 T. qingzhenensis Entire body Reduced 2.1–4.4 25.1–30.4 Truncated Absent Absent Complete Degenerated iii, 7–8 iii, 5 i, 8–9 i, 5 14 No Yes 4 + 36
25 T. qini Absent Absent Absent – Emarginated Present Absent Complete – ii, 8 ii, 5 – – 14–16 Yes No 4 + 34–35
26 T. qiubeiensis Absent Absent Absent – Emarginated – Absent Complete Degenerated iii, 7 iii, 5 i, 7–9 i, 5 14–15 Yes No 4 + 35
27 T. rongduensis Entire body Normal 7.2–14.7 24.1–28.6 Truncated Absent Absent Complete Degenerated iv, 9 iii, 5 i, 10 ii, 7 16 No Yes 4 + 39
28 T. rosa Absent Absent Absent – Emarginated Absent Absent Complete – iii, 9 iii, 6 i, 12 i, 7 14 Yes Yes –
29 T. sanduensis Entire body Normal 11.9–15.4 31.2–40.2 Emarginated Present Present Complete Degenerated ii, 8–9 i, 5 i, 8–9 i, 5 17–18 No Yes 4 + 37
30 T. shilinensis Absent Absent Absent – Truncated – Absent Complete Degenerated iii, 7 iii, 5 i, 8–10 i, 6 14 No Yes –
31 T. tianeensis Entire body Reduced 3.0–5.9 21.3–25.6 Truncated Present Absent Complete Degenerated iii, 6–7 iii, 6 i, 8–9 i, 5–6 15–16 No Yes 4 + 35
32 T. tianlinensis Absent Reduced Absent Absent Truncated Present Absent Complete Degenerated iv, 8–9 iii, 6 i, 10 i, 6 15–16 Yes Yes –
33 T. tianxingensis Entire body Normal 4.2–6.7 17.4–24.0 Truncated Absent Absent Complete Developed iii, 8 ii, 5 i, 9 i, 5 16 No No 4 + 38
34 T. wudangensis Entire body Reduced 5.1–6.5 33.1–35.8 Truncated Absent Absent Complete Degenerated iii, 7 iii, 5 i, 8 i, 5 14 No Yes 4 + 34
35 T. wulongensis Entire body Normal 11.1–19.1 38.5–43.1 Emarginated – Absent Complete Degenerated ii, 8–9 i, 5–6 i, 8–9 i, 5–7 18 No Yes 4 + 38–39
36 T. xiangshuingensis Entire body Normal 7.5 32.3 Emarginated – Absent Complete Degenerated iii, 6 iii, 5 i, 9 i, 6 14 No Yes –
37 T. xiangxiensis Absent Absent Absent – Emarginated – Absent Complete Developed iii, 8 iii, 6 i, 11 i, 6 16 Yes Yes –
38 T. xichouensis Entire body Reduced Absent – Truncated Absent Absent Complete Developed iii, 8 ii, 6 i, 9–10 i, 5–6 16 Yes Yes 4 + 36
39 T. xuanweiensis Absent Absent Absent – Emarginated – Absent Complete Well developed iii, 7–8 iii, 5 i, 10–12 i, 7–8 17–18 Yes No –
40 T. yunnanensis Entire body Normal 7.2–8.3 27.0–27.8 Emarginated Present Present Complete Degenerated iii, 7 iii, 5 i, 9–10 i, 7 15–16 No Yes –
41 T. zhenfengensis Entire body Normal 7.1–16.7 22.2–34.5 Truncated – Present Complete Degenerated iii, 7 iii, 5 i, 9 i, 5–7 14–15 No No 4 + 36

In August and December 2023, we collected several specimens of Triplophysa, identified by the closely set anterior and posterior nostrils, while conducting a survey of cave fishes in western Guizhou Province, China. Morphological examination and molecular phylogenetic analysis indicated that these specimens were distinct from the 39 hypogean species of Triplophysa. We formally describe two new species, Triplophysa ziyunensis sp. nov., and Triplophysa yaluwang sp. nov., based on evidence from morphology, mitochondrial, and nuclear genes.

Materials and methods

Sampling

Thirty-seven samples of six species were collected in total for morphology comparison and genetic analysis (Fig. 1). Of these five specimens representing the new species, we collected Triplophysa ziyunensis sp. nov., from Maoying Town, Ziyun County, Guizhou, and five specimens representing the new species, Triplophysa yaluwang sp. nov., from Maoying Town, Ziyun County, Guizhou. The following specimens were used for morphometric data: nine specimens were T. rosa from Huolu Town, Wulong County, Chongqing; three specimens were T. wudangensis from Wudang District, Guiyang City, Guizhou; eight specimens were T. qingzhenensis from Qingzhen County, Guiyang City, Guizhou; and seven specimens were T. guizhouensis from Baijin Town, Huishui County, Guizhou. All of the specimens were fixed in 10% buffered formalin and later transferred to 75% ethanol for preservation. Muscle samples used for molecular analysis were preserved in 95% alcohol and stored at −20 °C. All of the specimens were deposited at Guizhou Normal University (GZNU), Guiyang City, Guizhou Province, China.

DNA extraction, PCR, and sequencing

Genomic DNA was extracted from muscle tissue using a DNA extraction kit from Tiangen Biotech (Beijing) Co. Ltd. In total, six tissue samples used for molecular analysis were amplified and sequenced for mitochondrial gene cytochrome b (Cyt b) using the primers L3975 (5’-CGCCTGTTTACCAAAAACAT-3’) and H4551 (5’-CCGGTCTGAACTCAGATCACGT-3’) following Xiao et al. (2001). We also amplified one nuclear gene recombinase-activating 1 protein gene (RAG1) for 16 tissue samples, using primer LTF1 (5’-ATCATCGATGGCCTCTCAGGTT-3’) and LTR1 (5’-ACGTGGGCTAGAGTCTTGTGTAGGT-3’). PCR amplifications were performed within a 20 μl reaction volume with the cycling conditions that follow: An initial denaturing step at 95 °C for 4 min, 35 cycles of denaturing at 95 °C for 30 s, annealing at 45 °C (for Cyt b)/ 52 °C (for RAG1) for 40 s, and extension at 72 °C for 1 min followed by a final extension at 72 °C for 10 min. PCR products were purified with spin columns. The products were sequenced on an ABI Prism 3730 automated DNA sequencer at Chengdu TSING KE Biological Technology Co. Ltd. (Chengdu, China). All of the newly obtained sequences were submitted to GenBank (Table 3).

Table 3.
Download as
CSV
XLSX

Localities, voucher information, and GenBank numbers for all samples used. Numbers in bold were generated in this study.

ID Species Localities (* type localities) Voucher ID Cytb RAG1
1 T. guizhouensis Lewang Town, Wangmo County, Guzihou, China GZNU20220313001 OQ241174 PQ117091
2 T. guizhouensis Lewang Town, Wangmo County, Guzihou, China gznu09 KU987438 PQ117092
3 T. guizhouensis Baijin Town, Huishui County, Guzihou, China* GZNU20230722007 GZ01 PQ117093
4 T. yaluwang sp. nov. Maoying Town, Ziyun City, Guizhou, China* GZNU20240118005 PQ117067 PQ117090
5 T. yaluwang sp. nov. Maoying Town, Ziyun City, Guizhou, China* GZNU20240118006 PQ117068 PQ117089
6 T. longliensis / SWU2016090300 MW582825
7 T. sanduensis Zhonghe Town, Sandu County, Guizhou, China* SWU20170613001 MW582822
8 T. qini Houping Village, Wulong County, Chongqing, China* ON528184
9 T. xiangxiensis Feihu Cave, Hunan, China* / JN696407
10 T. xiangxiensis / IHB 2015010002 KT751089
11 T. nandanensis Hechi City, Guangxi,China SWU20151123046 MG697588
12 T. nandanensis Liuzhai Town, Nandan County, Guangxi, China* GZNU20230404005 OQ754126
13 T. nandanensis Liuzhai Town, Nandan County, Guangxi, China* GZNU20230404007 OQ754128
14 T. tianeensis / / MW582826
15 T. tianeensis Bala Township, Tian ‘e County, Guangxi, China* GZNU20230404003 OQ754124
16 T. nasobarbatula Dongtang Township, Libo County, Guizhou, China* GZNU20190114001 MH685911
17 T. nasobarbatula Dongtang Township, Libo County, Guizhou, China* GZNU20220313010 OQ241175
18 T. nasobarbatula Dongtang Township, Libo County, Guizhou, China* GZNU20220313011 OQ241176
19 T. macrocephala Lihu Town, Nandan County, Guangxi, China* GZNU20230404002 OQ754123
20 T. rosa Huolu Town, Wulong County, Chongqing, China* SWU10100503 JF268621
21 T. rosa / F3911 MG697587
25 T. rosa HuoLuTown, Wulong County, Chongqing City, China* GZNU20230404009 OQ754130 PQ117076
22 T. rosa Huolu Town, Wulong County, Chongqing, China* PQ117079
23 T. rosa Huolu Town, Wulong County, Chongqing, China* PQ117080
24 T. rosa Huolu Town, Wulong County, Chongqing, China* PQ117081
26 T. qingzhenensis Qingzhen County, Guiyang City, Guizhou, China* IHB 201911150004 MT700458
27 T. qingzhenensis Qingzhen County, Guiyang City, Guizhou, China* PQ117082
28 T. qingzhenensis Qingzhen County, Guiyang City, Guizhou, China* PQ117083
29 T. qingzhenensis Qingzhen County, Guiyang City, Guizhou, China* PQ117084
30 T. wudangensis Wudang District, Guiyang City, Guizhou, China* IHB 201908090003 MT700460
31 T. wudangensis Wudang District, Guiyang City, Guizhou, China* GZNU20230404010 OQ754131 PQ117085
32 T. wudangensis Wudang District, Guiyang City, Guizhou, China* PQ117086
33 T. wudangensis Wudang District, Guiyang City, Guizhou, China* PQ117087
34 T. wudangensis Wudang District, Guiyang City, Guizhou, China* PQ117071
35 T. ziyunensis sp. nov. Maoying Town, Ziyun City, Guizhou, China* GZNU20230529003 PQ117069 PQ117072
36 T. ziyunensis sp. nov. Maoying Town, Ziyun City, Guizhou, China* GZNU20230529004 PQ117069 PQ117073
37 T. ziyunensis sp. nov. Maoying Town, Ziyun City, Guizhou, China* GZNU20230529005 PQ117071 PQ117074
38 T. ziyunensis sp. nov. Maoying Town, Ziyun City, Guizhou, China* PQ117075
39 T. erythraea Dalong Cave, Huayuan County, Hunan, China* / MG967615
40 T. xuanweiensis Tangtang Town, Xuanwei City, Yunnan, China* ASIZB223818 OL675196
41 T. xuanweiensis Tangtang Town, Xuanwei City, Yunnan, China* ASIZB223819 OL675197
42 T. xuanweiensis Tangtang Town, Xuanwei City, Yunnan, China* ASIZB223820 OL675198
43 T. zhenfengensis Xinlongchang Town, Xingren City, Guizhou, China* GZNU20220313007 OQ241177
44 T. zhenfengensis Xinlongchang Town, Xingren City, Guizhou, China* GZNU20220313008 OQ241178
45 T. zhenfengensis Xinlongchang Town, Xingren City, Guizhou, China* GZNU20220313009 OQ241179
46 T. zhenfengensis Xinlongchang Town, Xingren City, Guizhou, China* GZNU20220313005 OQ241180
47 T. rongduensis Rongbei Town, Ceheng County, Guzihou, China* GZNU20230110001 OQ754135
48 T. rongduensis Rongbei Town, Ceheng County, Guzihou, China* GZNU20230110002 OQ754136
49 T. rongduensis Rongbei Town, Ceheng County, Guzihou, China* GZNU20230110003 OQ754137
50 T. anlongensis Xinglong Town, Anlong County, Guzihou, China* GZNU20230112001 OQ754138
51 T. anlongensis Xinglong Town, Anlong County, Guzihou, China* GZNU20230112002 OQ754139
52 T. anlongensis Xinglong Town, Anlong County, Guzihou, China* GZNU20230112003 OQ754140
53 T. baotianensis Baotian Town, Panzhou City, Guzihou, China* GZNU20180421005 MT992550
54 T. baotianensis Baotian Town, Panzhou City, Guzihou, China* GZNU20180421006 OQ241181
55 T. panzhouensis Hongguo Town, Panzhou City, Guizhou, China* GZNU20220513001 OQ754119
56 T. panzhouensis Hongguo Town, Panzhou City, Guizhou, China* GZNU20220513002 OQ754120
57 T. panzhouensis Hongguo Town, Panzhou City, Guizhou, China* GZNU20220513003 OQ754121
58 T. cehengensis Rongbei Town, Ceheng County, Guzihou, China* GZNU20230109001 OQ754132
59 T. cehengensis Rongbei Town, Ceheng County, Guzihou, China* GZNU20230109002 OQ754133
60 T. cehengensis Rongbei Town, Ceheng County, Guzihou, China* GZNU20230109003 OQ754134
61 T. huapingensis / F3917 MG697589
62 T. huapingensis Huaping Town, Leye County, Guangxi, China* GZNU20230404004 OQ754125
63 T. langpingensis Longping Township, Tianlin County, Guangxi* GZNU20230404001 OQ754122
64 T. qiubeiensis NijiaoVillage, Qiubei County, Yunnan , China* GZNU20230404006 OQ754127
65 T. wulongensis Wulong County, Chongqing, China* / MW582823
66 T. wulongensis HuoLuTown, Wulong County, Chongqing City, China GZNU20230404008 OQ754129
67 T. nujiangensa Fugong County, Yunnan, China IHB201315814 KT213598
68 T. tibetana Mafamu lake, Xinjiang, China NWIPB1106069 KT224364
69 T. tenuis Niutou river, Qingshui County, Gansu, China IHB0917490 KT224363
70 T. wuweiensis Yongchang County, Gansu, China IHB201307124 KT224365
71 Barbatula barbatula / / KP715096
72 Barbatula labiata Xinyuan County, Xinjiang, China IHB201306569 KT192057
73 Homatula berezowskii Qujing City, Yunnan, China FS-2014-Y03 NC_040302

Phylogenetic analyses and nuclear haplotyping

Sixty-three mitochondrial Cyt b sequences, including six newly sequenced and 57 downloaded from GenBank, were used for molecular analysis. We followed the phylogenetic study of Luo et al. (2023) and used Barbatula labia, B. barbatula, and Homatula berezowskii as outgroups (Table 3).

All of the sequences were assembled and aligned using the MUSCLE (Edgar 2004) module in MEGA v. 7.0 (Kumar et al. 2016) with default settings. Alignment results were checked visually. Phylogenetic trees were constructed via both maximum likelihood (ML) and Bayesian inference (BI) methods. The ML was conducted in IQ-TREE v. 2.0.4 (Nguyen et al. 2015) with 10,000 ultrafast bootstrap (UBP) replicates (Hoang et al. 2018), and it was performed until a correlation coefficient of at least 0.99 was reached. The BI was performed in MrBayes v. 3.2.1 (Ronquist et al. 2012), and the best-fit model was obtained based on the Bayesian information criterion computed with PartitionFinder v. 2.1.1 (Lanfear et al. 2017). The first, second, and third codons of Cyt b were defined in this analysis.

The analysis suggested the best partition scheme for each codon position of Cyt b. TRNEF+I+G, HKY+I, and TIM+I+G were selected for the first, second, and third codons, respectively. Two independent runs were conducted in the BI analysis, each of which was performed for 2× 107 generations and sampled every 1000 generations. The first 25% of the samples were discarded as a burn-in, resulting in a potential scale reduction factor of < 0.01. Nodes in the trees were considered well-supported when Bayesian posterior probabilities (BPP) were ≥ 0.95 and the ML ultrafast bootstrap value (UBP) was ≥ 95%. Uncorrected p-distances (1000 replicates) based on Cyt b were estimated using MEGA v. 7.0.

We also used the nuclear gene (RAG1) in PopART v. 1.7 (Leigh and Bryant 2015) based on the Median Joining method (Bandelt et al. 1999) to obtain haplotypes for assessing differences between the new species and genetically similar species.

Morphometrics, comparisons, and statistics

Morphometric data were collected from 37 well-preserved specimens of Triplophysa (Suppl. material 1). Twenty measurements were recorded to the nearest 0.1 mm with digital calipers following the protocols of Tang et al. (2012) and Li et al. (2018). All of the measurements were taken on the left side when looking directly at the head end of the fish.

Comparative data for the 39 hypogean species of Triplophysa were obtained from the literature and specimen examination (Table 2). Specimens of 19 species from the type locality were collected and examined, and these included: T. anlongensis, T. cehengensis, T. baotianensis, T. erythraea, T. guizhouensis, T. huapingensis, T. langpingensis, T. macrocephala, T. nasobarbatula, T. nandanensis, T. panzhouensis, T. qingzhenensis, T. qini, T. qiubeiensis, T. rosa, T. rongduensis, T. tianeensis, T. wudangensis, and T. zhenfengensis (see Suppl. material 1). The measurements of these species were also included in the statistical analysis, taking into consideration the morphological similarity, genetic differences, and geographical distances of the two new species to T. rosa, T. qingzhenensis, T. wudangensis, T. guizhouensis, T. sanduensis, and T. longliensis.

Principal component analyses (PCAs) of size-corrected measurements and simple bivariate scatterplots were used to characterize the morphometric differences between the new species and closely related species. Mann–Whitney U tests were used to determine the significance of differences in morphometric characteristics between the new species and similar species. All of the statistical analyses were performed using SPSS 21.0 (SPSS, Inc., Chicago, IL, USA), and differences were considered statistically significant at P < 0.05. PCAs of morphological data were performed after logarithmic transformation and under nonrotational conditions. All of the pre-processing of morphological data was performed in Microsoft Excel (Microsoft Corporation 2016).

Results

Phylogenetic analyses, genetic divergence, and nuclear haplotypes

ML and BI phylogenies were constructed based on mitochondrial Cyt b, with the sequence length being 1140 base pairs. The BI and ML phylogenetic trees showed a highly consistent topology that strongly supported the monophyly of the genus Triplophysa, and indicated that Triplophysa could be divided into two major clades, namely, the hypogean group and the epigean group (Fig. 2A).

Figure 2. 

Phylogeny and nuclear gene haplotypes A phylogenetic tree based on mitochondrial Cyt b (1140 bp). Bayesian posterior probabilities (BPP) from BI analysis/ultrafast bootstrap supports (UBP) from ML analysis are noted beside nodes. Scale bars represent 0.05 nucleotide substitutions per site. The numbers at the tips of species name correspond to the ID numbers listed in Table 2 B haplotypes inferred based on the nuclear gene RAG1.

The hypogean group contains 24 species from the karsts of southwest China (Chongqing, Guangxi, Guizhou, Hubei, and Yunnan) and two other lineages from western Guizhou that can be further divided into three clades (Fig. 2A): Clade A, only T. wulongensis, mainly in the Wujiang River basin (Fig. 1); subclade B1, including T. qiubeiensis, T. langpingensis, T. huapingensis, T. cehengensis, T. panzhouensis, T. baotianensis, T. anlongensis, T. rongduensis, and T. zhenfengensis, mainly in the Nampanjiang, Beipanjiang, and Hongshui River basins (Fig. 2A); and subclade B2 including T. xuanweiensis, T. erythraea, T. wudangensis, T. qingzhenensis, T. rosa, T. macrocephala, T. nasobarbatula, T. tianeensis, T. nandanensis, T. xiangxiensis, T. qini, T. sanduensis, T. longliensis, T. guizhouensis, and two other lineages from western Guizhou, mostly upstream of the Pearl and Yangtze rivers (Fig. 1).

All of the samples within subclade B1 from Shuitang Village, Maoying Town, Ziyun County, Guizhou Province (samples 35–38 in Table 3), clustered together in a sister clade to T. wudangensis, T. qingzhenensis, and T. rosa with strong node support (BPP/UBP = 1.00/1.00). This population could be distinguished from all of the known species and other undescribed lineages in this study via distinct morphological characteristics and molecular differences, with a lower p-distance of 1.8–2.0% (vs T. wudangensis, T. rosa, and T. qingzhenensis) (Table 4). Thus, the population at this locality represents an independently evolved lineage and is described below as a new species, Triplophysa ziyunensis sp. nov.

Table 4.
Download as
CSV
XLSX

Uncorrected p-distance (%) between new species and 24 congeneric species of the genus Triplophysa based on mitochondrial Cyt b.

ID Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
1 T. ziyunensis sp. nov.
2 T. yaluwang sp. nov. 9.5
3 T. anlongensis 15.3 14.5
4 T. baotianensis 14.9 14.0 6.8
5 T. cehengensis 15.1 13.8 3.8 6.8
6 T. erythraea 11.5 10.3 14.6 13.8 14.6
7 T. guizhouensis 9.8 1.4 14.7 14.5 13.9 10.6
8 T. huapingensis 15.1 14.1 9.9 10.2 9.3 14.9 14.6
9 T. langpingensis 14.2 14.7 14.1 14.0 13.8 15.8 15.1 14.4
10 T. longliensis 10.0 2.5 14.6 14.2 14.2 10.6 2.8 14.6 15.0
11 T. macrocephala 10.0 9.2 15.4 15.9 15.3 12.0 9.6 14.9 15.7 9.4
12 T. microphthalmus 14.5 13.8 10.0 11.0 9.5 15.1 13.8 5.9 13.8 14.0 15.1
13 T. nandanensis 10.9 9.7 16.5 16.3 16.0 12.3 10.1 16.0 16.4 10.3 5.3 16.0
14 T. nasobarbatula 9.9 9.1 15.5 15.8 15.2 12.1 9.6 14.9 15.3 9.4 0.9 15.2 5.5
15 T. panzhouensis 15.8 13.6 7.0 7.7 8.2 13.4 14.3 10.1 14.7 12.9 15.3 10.2 15.7 15.2
16 T. qingzhenensis 2.0 8.5 15.4 15.1 15.3 11.3 8.9 14.6 14.4 9.1 9.7 14.3 10.2 9.5 15.2
17 T. qini 9.5 5.1 14.7 15.5 14.5 10.7 5.0 15.1 15.6 5.3 9.1 13.9 10.5 9.4 14.3 8.8
18 T. qiubeiensis 14.1 12.7 14.7 14.2 14.2 13.8 12.8 14.6 14.5 13.3 14.6 14.5 14.5 14.2 14.4 13.8 13.4
19 T. rosa 1.9 8.7 15.4 15.1 15.4 11.6 9.0 15.3 14.5 9.3 10.0 14.6 10.7 9.7 15.5 1.4 9.1 13.9
20 T. sanduensis 9.9 2.5 14.8 14.7 14.5 11.0 2.6 14.7 15.1 0.7 9.3 13.9 10.5 9.2 13.5 9.0 5.3 13.6 9.3
21 T. tianeensis 10.6 9.8 16.6 16.5 16.5 11.6 10.2 16.1 16.2 10.5 5.1 16.3 2.0 5.3 15.8 10.0 10.0 14.6 10.6 10.6
22 T. wudangensis 1.8 8.7 15.3 14.9 15.4 11.3 9.1 14.8 14.4 9.3 10.1 14.4 10.7 9.8 15.5 1.6 9.3 14.2 1.5 9.3 10.5
23 T. wulongensis 13.7 14.1 16.9 16.6 16.5 15.4 13.9 17.7 15.5 13.5 15.1 16.5 14.8 15.1 16.3 13.6 13.4 15.5 13.8 13.4 14.6 13.8
24 T. xiangxiensis 9.3 7.9 14.3 14.7 14.2 11.2 8.1 15.2 14.6 7.2 8.5 14.5 9.7 8.4 13.9 8.6 5.9 13.8 8.7 7.8 9.0 9.0 14.4
25 T. xuanweiensis 11.3 11.1 14.8 14.2 14.8 11.9 11.6 14.4 14.0 11.8 11.5 14.5 11.7 11.4 14.1 11.5 11.4 12.3 11.6 11.7 12.0 11.2 14.2 11.4
26 T. zhenfengensis 15.6 13.9 3.4 6.8 0.9 14.5 14.0 9.0 13.6 14.4 15.6 9.3 16.2 15.3 7.7 15.4 14.8 14.1 15.6 14.6 16.7 15.5 16.3 14.3 14.4

All of the samples within subclade B1 from Xinzhai Village, Maoying Town, Ziyun County, Guizhou Province (samples 4 and 5 in Table 3), clustered together in a sister clade to T. guizhouensis with strong node support (BPP/UBP = 0.98/0.96). This population could be distinguished from all of the known species and other undescribed lineages in this study by distinct morphological characteristics and molecular differences, with a lower p-distance of 1.4% (vs T. guizhouensis) (Table 4). Thus, the population at this locality represents an independently evolved lineage and is described below as a new species, Triplophysa yaluwang sp. nov.

Haplotype networks based on RAG1 showed that unique, non-shared haplotypes were observed in the two new species and multiple linking mutations occurred with closely related species (Fig. 2B). We observed shared haplotypes from among T. qingzhenensis, T. rosa, and T. wudangensis (Fig. 2B). More haplotype diversity was found within T. rosa, a pattern that may be related to higher genetic diversity and wider distribution.

Morphological analyses

Mann-Whitney U tests revealed differences in several morphological characteristics among the two new species (T. ziyunensis sp. nov. and T. yaluwang sp. nov.), and between the new species and the closely related species (Table 5). These significantly different measurements were concentrated on the head, barbel, fins, and tail (Table 5). There are significant morphological differences only in eye diameter and pectoral-fin ray length for Triplophysa yaluwang sp. nov. and T. guizhouensis.

Table 5.
Download as
CSV
XLSX

Morphological comparison of Triplophysa ziyunensis sp. nov. (TZ), Triplophysa yaluwang sp. nov. (TY), T. wudangensis (TW), T. rosa (TR), T. qingzhenensis (TQ), and T. guizhouensis (TG). All units in mm. P-values are at the 95% significance level.

T. ziyunensis sp. nov. T. yaluwang sp. nov. T. wudangensis T. rosa T. qingzhenensis T. guizhouensis TY vs TG TZ vs TW TZ vs TR TZ vs TQ
Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD
Total length 78.6–120.0 103.8 ± 16.3 66.5–99.4 77.4 ± 15.3 73.4–85.9 79.5 ± 6.3 62.3–130.8 91.8 ± 23.8 84.6–123.3 109.6 ± 14.0 54.9–88.1 75.5 ± 12.2 0.808 0.101 0.386 0.38
Standard length 63.3–100.1 85.2 ± 14.4 54.1–83.9 64.3 ± 13.7 59.8–66.8 63.7 ± 3.6 49.8–104.3 74.0 ± 19.0 72.2–103 91.7 ± 11.4 45.8–72.8 62.5 ± 9.9 0.935 0.101 0.317 0.38
Head length 17.4–26.2 22.5 ± 3.6 13.4–19.7 15.3 ± 2.8 11.5–12.9 12.3 ± 0.7 14.7–28.6 21.0 ± 4.8 15.8–24.4 21.2 ± 3.2 6.2–16.7 13.3 ± 3.7 0.372 0.025 0.463 0.464
Head depth 8.6–11.9 10.5 ± 1.5 6.1–9.8 7.4 ± 1.7 6.6–7.4 7.0 ± 0.4 6.8–16.1 10.4 ± 3.0 8.6–12.9 11.3 ± 1.7 5.8–8.3 7.2 ± 0.9 0.935 0.025 0.739 0.188
Head width 10.5–16.1 12.9 ± 2.6 6.9–12.4 9.0 ± 2.4 8.2–9.2 8.8 ± 0.5 8.8–17.2 11.8 ± 2.6 10.2–15.8 13.5 ± 2.0 7.1–10.9 9.4 ± 1.3 0.57 0.025 0.386 0.884
Snout length 8.6–12 10.4 ± 1.6 0.0–9.3 6.0 ± 3.7 6.2–7 6.6 ± 0.4 7.3–10.4 8.7 ± 1.3 7.9–12.3 10.7 ± 1.7 5.2–7.4 6.4 ± 0.8 0.935 0.025 0.142 0.941
Eye diameter 0.4–1.2 0.9 ± 0.3 0.0–1.1 0.7 ± 0.4 0.6–0.7 0.6 ± 0.1 0.0–0.4 0.0 ± 0.1 0.2–0.3 0.2 ± 0.1 1.7–2.3 1.9 ± 0.2 0.004 0.227 0.001 0.003
Interorbital distance 4.5–6.5 5.4 ± 0.9 0.0–5 3.2 ± 1.9 4.0–4.5 4.3 ± 0.2 4.7–6.8 5.7 ± 0.9 5.1–7.7 6.8 ± 1.0 2.1–4.3 3.3 ± 0.9 0.685 0.025 0.327 0.028
Body depth 9.4–14.7 12.2 ± 2.3 6.5–13.5 9.1 ± 2.8 6.6–7.3 7.0 ± 0.4 5.9–17.2 10.2 ± 3.9 11.7–17 14.8 ± 1.9 6.3–11.2 8.8 ± 1.7 0.935 0.025 0.162 0.057
Body width 7.6–13.2 10.6 ± 2.5 8.1–9.1 8.5 ± 0.4 4.8–5.4 5.1 ± 0.3 4.3–14.7 7.9 ± 3.4 10.6–15.6 13.3 ± 1.9 5.8–9.8 7.3 ± 1.4 0.062 0.025 0.096 0.057
Maxillary barbel length 6.0–9.5 8.5 ± 1.5 3.0–6.8 4.9 ± 1.4 5.2–5.9 5.6 ± 0.3 5.1–10.3 7.5 ± 2.0 5.5–9.5 7.6 ± 1.3 3.9–6.4 5.2 ± 0.8 0.745 0.025 0.549 0.188
Outrostral barbel length 10.1–14.5 12.1 ± 1.8 5.3–8.1 6.4 ± 1.3 7.2–8.0 7.6 ± 0.4 6.5–10.6 8.4 ± 1.5 6.7–10.6 8.9 ± 1.3 4.6–7.7 6.2 ± 1.0 0.935 0.025 0.006 0.008
Inrostral barbel length 4.6–7 5.7 ± 0.9 2.2–4.5 3.5 ± 1.0 3.1–3.5 3.3 ± 0.2 3.2–6.5 4.5 ± 1.1 3.8–5.4 4.7 ± 0.6 2.8–3.9 3.3 ± 0.4 0.465 0.025 0.039 0.028
Dorsal-fin length 15.0–22.6 19.2 ± 3.2 11.1–16.1 13.0 ± 2.3 28.3–31.7 30.2 ± 1.7 13.4–26.9 18.2 ± 4.3 12.3–20.1 16.8 ± 2.9 9.0–14.4 12.6 ± 1.8 0.935 0.025 0.463 0.242
Dorsal-fin base length 9.6–13.4 11.8 ± 1.6 5.9–10.3 7.8 ± 1.8 7.3–8.2 7.8 ± 0.5 7.4–15.8 10.7 ± 3.2 7.9–9.6 8.8 ± 0.6 5.6–9.5 8.1 ± 1.3 0.808 0.025 0.463 0.005
Pectoral-fin length 13.7–21.7 18.5 ± 3.0 11.3–16.8 13.6 ± 2.2 12–13.4 12.8 ± 0.7 14.4–35.9 21.0 ± 6.6 14.3–21.7 17.9 ± 2.6 7.4–12.2 10.3 ± 1.6 0.019 0.025 0.386 0.661
Anal-fin length 11.9–18.2 15.6 ± 2.6 9.2–13.5 10.7 ± 1.9 9.8–10.9 10.4 ± 0.6 9.5–25.3 15.8 ± 4.8 10.2–16.1 14.0 ± 2.0 8.0–11.5 10.2 ± 1.2 0.935 0.025 0.841 0.188
Pelvic-fin length 11.6–19.3 15.3 ± 2.8 9.0–13.3 10.5 ± 1.9 9.2–10.2 9.8 ± 0.5 8.9–24.4 15.1 ± 4.8 10.8–16.8 14.0 ± 2.1 9.0–14.7 12.0 ± 1.8 0.372 0.025 0.641 0.38
Caudal peduncle length 9.8–16.9 13.6 ± 3.0 9.8–16.5 12.0 ± 2.8 11.8–13.1 12.5 ± 0.7 8.5–17.7 11.9 ± 3.5 11.9–18.2 15.9 ± 2.4 6.6–12.8 9.9 ± 2.4 0.291 0.655 0.257 0.107
Caudal peduncle depth 4.8–6.7 5.8 ± 0.8 3.8–6.6 4.8 ± 1.2 3.9–4.4 4.2 ± 0.2 3.1–9.3 5.5 ± 2.2 6.5–9.4 7.9 ± 1.0 3.5–5.9 4.8 ± 1.0 0.871 0.025 0.205 0.005

Four principal component factors with eigenvalues greater than one were extracted based on the PCA of the morphological data. These factors accounted for 83.42% and 74.86% of the total variation (Suppl. material 2). The first principal component (PC1) accounted for 38.23% and 28.77% of the variation and was positively correlated with all of the variables (eigenvalue = 3.0 and 4.1). On the two-dimensional plots of PC1 and PC2, the new species T. ziyunensis sp. nov. can be readily distinguished from T. wudangensis, T. rosa, and T. qingzhenensis (Fig. 3A). T. yaluwang sp. nov. can be readily distinguished from T. guizhouensis (Fig. 3B), while the holotype of T. longliensis are mosaic in the T. yaluwang sp. nov. The two new species are clearly distinguished by morphological characteristics from the geographical and morphological relative species based on statistical analysis of the measurements and the PCA result.

Figure 3. 

Plots of principal components analysis scores of A T. ziyunensis sp. nov. and B T. yaluwang sp. nov., and closely related species based on morphometric data.

Taxonomic account

Triplophysa ziyunensis Wu, Luo, Xiao & Zhou, sp. nov.

https://zoobank.org/BA4F7B39-A976-4D59-AE66-439DB9130A2E
Figs 4, 5, Table 5, Suppl. material 1

Type material

Holotype. GZNU20230529001 (Fig. 4), 105.1 mm total length (TL), 86.7 mm standard length (SL), collected by Li Wu and Xing-Liang Wang on 29 May 2023, at Shuitang Village, Maoying Town, Ziyun County, Guizhou Province, China (25.96846238°N, 106.13737106°E; 1228 m a.s.l.; Fig. 1).

Figure 4. 

Morphological characteristics of holotype GZNU20230529001 of Triplophysa ziyunensis sp. nov. in preservative (10% formalin) A dorsal view B ventral view C lateral view D dorsal view of head E lateral view of head, and F ventral view of head.

Paratypes. Four specimens from the same locality as the holotype: GZNU20230226008-226010, and GZNU20230529002, 63.3–100.1 mm SL, collected by Tao Luo, Li Wu, Xing-Liang Wang, Xin-Rui Zhao, and Chang-Ting Lan on 26 February 2023.

Diagnosis

Triplophysa ziyunensis sp. nov. is distinguished from other hypogean species of the genus Triplophysa by the following characteristics in combination: (1) body naked, scaleless, pigmented markings on surface of body, except ventral; (2) eyes reduced, diameter 2.4–4.9% of head length (HL); (3) pelvic-fin tip extending to anus; (4) tip of pectoral fin not reaching pelvic fin origin; (5) anterior and posterior nostrils closely set, with anterior nostril elongated to a barbel-like tip; (6) tip of outrostral barbel extending backward, not reaching anterior margin of eye; (7) lateral line complete; (8) posterior chamber of air bladder degenerated; and (9) dorsal-fin rays iii-8, pectoral-fin rays i-10, pelvic-fin rays i-6, anal-fin rays iii-5, and 16 branched caudal-fin rays.

Description

Morphological data on the specimens of Triplophysa ziyunensis sp. nov. are provided in Table 5 and Suppl. material 1. Body elongated and cylindrical, posterior portion gradually compressed from dorsal fin to caudal-fin base, with deepest body depth anterior to dorsal-fin origin, deepest body depth 13–16% of standard length (SL). Dorsal profile slightly convex from snout to dorsal-fin insertion, and then straight from posterior portion of dorsal-fin origin to caudal-fin base. Ventral profile flat. Head short, length 26–27% of SL, slightly depressed and flattened, width slightly greater than depth (head width (HW)/head depth (HD) = 1.1–1.3). Snout slightly pointed, and snout length 46–50% of HL. Mouth inferior and curved, mouth corner situated below anterior nostril, upper and lower lips smooth, lower lip with V-shaped median notch. Three pairs of barbels are present: inner rostral barbel long, length 23–27% of HL, backward extending to corner of the mouth; out rostral barbel long, length 52–58% of HL, backward extending to beyond posterior margin of eyes. Maxillary barbel not extending to posterior margin of operculum, length 34–42% of HL. Anterior and posterior nostrils closely set, length 0.20–0.25 mm. Anterior nostril tube long, with an elongated short barbel-like tip, tip of posterior nostril extending backward not reaching to anterior margin of the eye. Eyes reduced, with diameter 2–5% of HL. Gill opening small, gill rakers not developed, ten inner gill rakers on first gill arch (n = 1).

Dorsal-fin rays iii-8, pectoral-fin rays i-10, pelvic-fin rays i-6, anal-fin rays i-5, 16 branched caudal-fin rays. Dorsal fin short, length 20–23% of SL, distal margin emarginated, origin anterior to pelvic-fin insertion and situated slightly posterior to the midpoint between snout tip and caudal-fin base, first branched ray longest, shorter than head length, tip of dorsal fin vertical to the anus. Pectoral fin moderately developed, length 22–24% of SL, tip of pectoral fin extending backward almost to midpoint between origin of pectoral and pelvic fin origins, not reaching to pelvic fin origin. Pelvic fin length 16–20% of SL, vertically aligned with third branched ray of dorsal fin, tips of pelvic fin reaching anus. Anal fin length 16–20% of SL, distal margin truncated, origin close to anus, tips of anal fin not reaching caudal-fin base, distance between tips of anal fin and anus 8.5× the eye diameter. Caudal fin forked, upper lobe equal in length to lower lobe, tips pointed, caudal peduncle length ~ 13.6 mm, caudal peduncle depth ~ 5.8 mm, with weak adipose crests along both dorsal and ventral sides. Total vertebrae: 39 (n = 1).

Cephalic lateral line system developed. Lateral line complete, exceeding tip of pectoral fin and reaching base of caudal fin. Two chambers of air bladder, anterior chamber dumbbell-shaped and membranous, open on both sides, slightly closed posteriorly; posterior chamber degenerated, slightly filling the body cavity, connected with anterior chamber by a long, slender tube.

Coloration

In cave water, the body of living fish is semi-translucent and pale pink, with irregular dark brownish brown patches on the head and body (Fig. 5). After fixation in 10% formalin solution, the body color was pale grey, and the dark-brown patches on the head and body were more prominent (Fig. 4).

Figure 5. 

Ecological photographs of Triplophysa ziyunensis sp. nov. and closely related species in life A Triplophysa ziyunensis sp. nov. B T. rosa C T. wudangensis, and D T. qingzhenensis, from Dr. Zhi-Xuan Zeng.

Secondary sex characteristics

No secondary sex characteristics were observed based on the present specimens of Triplophysa ziyunensis sp. nov.

Comparisons

Detailed comparative morphological data of Triplophysa ziyunensis sp. nov. with the 39 recognized hypogean species of Triplophysa are given in Table 2. Triplophysa ziyunensis sp. nov. is genetically close to T. qingzhenensis, T. rosa, and T. wudangensis and shares some similar morphological characters, such as reduced eye degeneration and degenerated body pigmentation, pigmented markings on the body surface, except ventral, but can still be distinguished by a combination of some morphological characters.

Triplophysa ziyunensis sp. nov. is be distinguished from T. qingzhenensis and T. wudangensis by having 10 branched pectoral fin rays (vs 8–9), 6 branched pelvic-fin rays (vs 5), 16 branched caudal fin rays (vs 14–15), and inhabiting the Pearl River basin (vs Yangtze River basin).

Triplophysa ziyunensis sp. nov. can be distinguished from T. rosa by having reduced body pigmentation, pigmented markings on body surface, except ventral (vs absence), eyes reduced, diameter 2.4–4.9% of HL (vs absent), 8 branched dorsal fin rays (vs 9), 10 branched pectoral fin rays (vs 12), 6 branched pelvic-fin rays (vs 7), 16 branched caudal fin rays (vs 14), and inhabiting the Pearl River basin (vs Yangtze River basin).

Ecology and distribution

Triplophysa ziyunensis sp. nov. has only been found in one cave in Shuitang Village, Maoying Town, Ziyun County, Guizhou Province, China, at an elevation of 1134 m. The pool where the new species was found is more than 15 m long, 13 m wide, and ~ 3 m deep, with a slow flow of water, and is located 80 m further inside the entrance of the cave. Inside the cave, another fish (Sinocyclocheilus multipunctatus, three individuals), bats (Rhinolophus sp., five individuals), and frogs (Odorrana wuchuanensis, 11 individuals) were found. Outside the cave, rapeseed and peppers were being grown. The population of the new species is very small and only five specimens were collected.

Remarks

The new species, Triplophysa ziyunensis sp. nov., inhabits the underground rivers of the type locality. Eyes are present and reduced, and with irregular dark brownish brown patches on the head and body. Therefore, this species can be considered as a stygophile fish within the hypogean group of the genus Triplophysa.

Etymology

The specific epithet ziyunensis refers to the type locality of the new species: Shuitang Village, Maoying Town, Ziyun County. We propose the common English name “Ziyun high-plateau loach” and the Chinese name “Zǐ Yún Gāo Yuán Qīu (紫云高原鳅)”.

Triplophysa yaluwang Lan, Liu, Zhou & Zhou, sp. nov.

https://zoobank.org/DE306B1B-F770-4E79-9B9E-400CEC202266
Figs 6, 7, Table 5, Suppl. material 1

Type material

Holotype. GZNU20240118001 (Fig. 6), 87.6 mm total length (TL), 73.9 mm standard length (SL), collected by Jia-Jun Zhou on 18 January 2024, in Xinzhai Village, Maoying Town, Ziyun County, Guizhou Province, China (25.89908752°N, 106.07921141°E, 1276 m a.s.l.; Fig. 1).

Figure 6. 

Morphological characteristics of holotype GZNU20240118001 of Triplophysa yaluwang sp. nov. in preservative (10% formalin) A dorsal view B ventral view C lateral view D dorsal view of head E lateral view of head, and F ventral view of head.

Paratypes. Four specimens from the same locality as the holotype: GZNU20240118002–118005, 54.1–83.9 mm SL, collected by Jia-Jun Zhou and Ye-Wei Liu on 27 September 2023.

Diagnosis

Triplophysa yaluwang sp. nov. is distinguished from other hypogean species of the genus Triplophysa by the following characteristics in combination: (1) body naked, scaleless, with irregular pale dark brownish brown markings, except ventral; (2) eyes reduced, diameter 4.6–6.1% of head length; (3) pelvic-fin tip reaching anus; (4) tip of pectoral fin not reaching to pelvic fin origin; (5) anterior and posterior nostrils closely set, with the anterior nostril elongated to a barbel-like tip; (6) tip of outrostral barbel extending backward, not reaching to anterior margin of eye; (7) lateral line complete; (8) posterior chamber of air bladder degenerated; and (9) dorsal-fin rays iii-7, pectoral-fin rays i-9, pelvic-fin rays i-5, anal-fin rays i-5, and 14 branched caudal-fin rays.

Description

Morphological data of Triplophysa yaluwang sp. nov. specimens are provided in Table 5 and Suppl. material 1. Body elongated and cylindrical, posterior portion gradually compressed from dorsal fin to caudal-fin base, with deepest body depth anterior to dorsal-fin origin, deepest body depth 12–16% of SL. Dorsal profile slightly convex from snout to dorsal-fin insertion, then straight from posterior portion of dorsal-fin origin to caudal-fin base. Ventral profile flat. Head short, length 26–27% of SL, slightly depressed and flattened, width slightly greater than depth (HW/HD = 1.1–1.3). Snout slightly pointed, and snout length 43–52% of HL. Mouth inferior and curved, mouth corner situated below anterior nostril, upper and lower lips smooth, lower lip with V-shaped median notch. Three pairs of barbels are present: inner rostral barbel long, length 16–27% of HL, backward extending to corner of mouth; out rostral barbel long, length 39–44% of HL, backward extending to beyond anterior margin of eyes. Maxillary barbel not extending to posterior margin of operculum, length 22–36% of HL. Anterior and posterior nostrils closely set, length 0.44–0.82 mm. Anterior nostril tube long, with an elongated short barbel-like tip, tip of posterior nostril extending backwards not reaching to anterior margin of eye. Eyes reduced, with diameter 5–6% of HL. Gill opening small, gill rakers not developed, nine inner gill rakers on first gill arch (n = 1).

Dorsal-fin rays iii-7, pectoral-fin rays i-9, pelvic-fin rays i-5–6, anal-fin rays i-5, 14 branched caudal-fin rays. Dorsal fin short, length 19–22% of SL, distal margin emarginated, origin anterior to pelvic-fin insertion and situated slightly posterior to the midpoint between snout tip and caudal-fin base, first branched ray longest, shorter than head length, tip of dorsal fin vertical to anus. Pectoral fin moderately developed, length 19–25% of SL, tip of pectoral fin extending backward almost to the midpoint between origin of pectoral and pelvic fin origins, not reaching to pelvic fin origin. Pelvic fin length 16–17% of SL, vertically aligned with second branched ray of dorsal fin, tips of pelvic fin reaching to anus. Anal fin length 16–18% of SL, distal margin truncated, origin close to anus, tips of anal fin not reaching caudal-fin base, distance between tips of anal fin and anus 2.2× the eye diameter. Caudal fin forked, upper lobe slightly longer than lower lobe, tips pointed, caudal peduncle length ~ 12 mm, caudal peduncle depth ~ 4.8 mm, with weak adipose crests along both dorsal and ventral sides. Total vertebrae: 40 (n = 1).

Cephalic lateral line system developed. Lateral line complete, exceeding tip of pectoral fin and reaching base of caudal fin. Two chambers of air bladder, anterior chamber dumbbell-shaped and membranous, open on both sides, slightly closed posteriorly; posterior chamber degenerated, slightly filling the body cavity, connected with anterior chamber by a long, slender tube.

Coloration

In cave water, living fish were semi-translucent with a pale pink body with irregular dark brownish brown patches on the Entire body (Fig. 7). After fixation in 10% formalin, the body color was white, and the dark brown color lightened (Fig. 6).

Figure 7. 

Ecological photographs of Triplophysa yaluwang sp. nov. and closely related species in life A Triplophysa yaluwang sp. nov. (paratype, GZNU20240118002) B Triplophysa yaluwang sp. nov. (paratype, GZNU20240118005), and C T. guizhouensis.

Variations

Among the five specimens collected, GZNU20240118002–118004 are essentially identical to the holotype in fin characteristics and body coloration. GZNU20240118005 differs from the holotype by the absence of body pigmentation and the absence of the eye (Fig. 7).

Secondary sex characteristics

Secondary sex characteristics were not observed in the specimens of Triplophysa yaluwang sp. nov.

Comparisons

Detailed morphological comparative data of Triplophysa yaluwang sp. nov. with Triplophysa ziyunensis sp. nov. and the 39 hypogean species of Triplophysa are given in Table 2. Triplophysa yaluwang sp. nov. is genetically close to T. guizhouensis, T. longliensis, and T. sanduensis, but it can be distinguished in combination with morphological characteristics.

Triplophysa yaluwang sp. nov. can be distinguished from Triplophysa ziyunensis sp. nov. by having dorsal fin distal margin being emarginated (vs truncated), total vertebrae 40 (vs 39), seven branched dorsal fin rays (vs 8), nine branched pectoral fin rays (vs 10), and 14 branched caudal fin rays (vs 16).

Triplophysa yaluwang sp. nov. is distinguished from T. longliensis by having eyes reduced, small diameter 4.6–6.1% of HL (vs normal, diameter 9.5–11.5% of HL), interorbital width, 24.3–26.0% of HL (vs 31.4–37.5 of HL), total vertebrae 40 (vs 42), degenerated posterior chamber of air bladder (vs developed), seven branched dorsal-fin rays (vs 8), nine branched pectoral-fin rays (vs 10), and 14 branched caudal-fin rays (vs 15–16).

Triplophysa yaluwang sp. nov. is distinguished from T. sanduensis by having eyes reduced, small diameter 4.6–6.1% of HL (vs normal, diameter 11.9–15.4% of HL), interorbital width, 24.3–26.0% of HL (vs 31.2–40.2 of HL), total vertebrae 40 (vs 41), dorsal-fin rays, iii, 7 (vs ii, 8–9), three unbranched anal-fin rays (vs 1), 14 branched caudal-fin rays (vs 17–18), and tip of pelvic fin reaching anus (vs not reaching anus).

Triplophysa yaluwang sp. nov. differs from T. guizhouensis by having eyes reduced, diameter 4.6–6.1% of HL (vs normal, diameter 9.4–12.1% of HL), dorsal fin distal margin being emarginated (vs truncated), body scaleless (vs body covered by sparse scales), degenerated posterior chamber of air bladder (vs developed), seven branched dorsal fin rays (vs 8), five branched anal-fin rays (vs 6), and tip of pelvic fin reaching anus (vs not reaching anus).

Ecology and distribution

The new species Triplophysa yaluwang sp. nov. was found in one cave far from the village of Xinzhai Village, Maoying Town, Ziyun County, Guizhou Province, China (Fig. 1), in a water system where the underground river is a tributary of the Hongshui River. The cave habitat is a vertical shaft with an entrance located halfway up the mountainside. The underground river is approximately 150 m deep from the entrance, and the accessible portion is around 200 m long, 3 m wide, and 1–2 m deep. In this cave, the new species is sympatric with Sinocyclocheilus multipunctatus and some unnamed spiders.

Remarks

The new species, Triplophysa yaluwang sp. nov., inhabits the underground rivers of the type locality. Eyes are present and reduced, and with irregular dark brownish brown patches on the head and body. Therefore, this species can be considered as a stygophile fish within the hypogean group of the genus Triplophysa.

Etymology

The specific epithet yaluwang comes from King Yalu, a hero to the Miao people of Ziyun County, Guizhou Province, China, where the type locality is found. He was the 18th generation leader of the Miao ancestors in western China and led the Miao people through many trials and tribulations. He eventually carved out a suitable land for his people to live in near the type locality. His deeds have been preserved in the form of a song, which has been organized into the first full-length heroic epic of the Hmong, King Yalu. We propose the common English name “King Yalu high-plateau loach” and the Chinese name “Yà Lǔ Wáng Gāo Yuán Qīu (亚鲁王高原鳅)”.

Discussion

We describe two new species, Triplophysa ziyunensis sp. nov. and Triplophysa yaluwang sp. nov., based on morphological comparisons (Table 2), mitochondrial DNA sequence differences, and nuclear gene haplotypes (Fig. 2). The description of these two new species increases the number of species in the hypogean group of Triplophysa from 39 to 41 and the previous number of known species from Guizhou is increased to 15. The previous 13 species are T. cehengensis, T. rongduensis, T. panzhouensis, T. anlongensis, T. baotianensis, T. guizhouensis, T. longliensis, T. nasobarbatula, T. qingzhenensis, T. sanduensis, T. wudangensis, T. wulongensis, and T. zhenfengensis (Table 1). Our previous studies revealed that the cave-dwelling species of Triplophysa in Guizhou are concentrated in the south, central, and southwest (Fig. 1). Before this study, a large recording gap existed between central-southern and western Guizhou (Fig. 1), suggesting the possible presence of cryptic species in this region (Luo et al. 2023). This hypothesis was supported by the results of the present study. Similarly, discontinuities with previous records remain in northeastern and eastern Guizhou, suggesting that it would be useful to focus efforts there on the discovery of new species or new distribution areas.

The new species described here have only slight mitochondrial differences from closely related species (Table 4). For example, the new species T. yaluwang sp. nov. clusters with T. guizhouensis, both from the Hongshui River drainage basin, at a genetic distance of 1.4%. The type locality of the two new species is near the watershed between the Pearl and Yangtze rivers, i.e., the Miaoling Mountains (Fig. 1). Geological evidence suggests that the Miaoling mountains formed in the Early Pleistocene and eventually became the watershed between the Pearl River and Yangtze River systems during the mid- to late Pleistocene (Zhou and Chen 1993). Thus, the slight mitochondrial differences are associated with multiple connectivity between rivers, includes both surface and underground rivers, which leads to potential mitochondrial introgression (Yuan et al. 2023). Similar gene flow was observed among species of Triplophysa in the Qinghai-Tibetan Plateau region (Feng et al. 2019). This hypothesis is also supported by the mitochondrial matrilineal tree in this study, i.e., the species of the independent hydrological origin are not clustered together in the phylogenetic tree, but rather are distributed in a mosaic fashion (Figs 1, 2). However, this could also be related to short-term radial species formation. To evaluate this situation, we suggest the use of additional nuclear genetic markers or genomic evidence in the description of new species.

Acknowledgments

We thank Hao Wang, Xing-Liang Wang, Wei-Feng Wang, Cui Fan, Zhi-Xia Chen, and others for help with specimen collections. We thank LetPub (www.letpub.com) for its linguistic assistance during manuscript preparation.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was supported by the the Guizhou Normal University Academic Emerging Talent Fund Project (Qianshi Xin Miao [2021] 20), and the programs of the Strategic Priority Research Program B of the Chinese Academy of Sciences (CAS) (No. XDB31000000).

Author contributions

All authors have contributed equally.

Author ORCIDs

Chang-Ting Lan https://orcid.org/0009-0007-2381-3601

Li Wu https://orcid.org/0000-0002-7898-7517

Tao Luo https://orcid.org/0000-0003-4186-1192

Ye-Wei Liu https://orcid.org/0000-0003-4712-1072

Jia-Jun Zhou https://orcid.org/0000-0003-1038-1540

Jing Yu https://orcid.org/0009-0004-3629-3826

Xin-Rui Zhao https://orcid.org/0000-0002-9125-6276

Ning Xiao https://orcid.org/0000-0002-7240-6726

Jiang Zhou https://orcid.org/0000-0003-1560-8759

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

References

  • Chen YR, Yang JX, Xu GC (1992) A new blind loach of Triplophysa from Yunnan stone forest with comments on its phylogenetic relationship. Zoological Research 13(1): 17–23. [In Chinese]
  • Chen XY, Cui GH, Yang JX (2004) A new cave dwelling fish species of genus Triplophysa (Balitoridae) from Guangxi, China. Zoological Research 25(3): 227–231. [In Chinese]
  • Chen S, Sheraliev B, Shu L, Peng Z (2021) Triplophysa wulongensis, a new species of cave-dwelling loach (Teleostei, Nemacheilidae) from Chongqing, Southwest China. ZooKeys 1026: 179–192. https://doi.org/10.3897/zookeys.1026.61570
  • Chu XL, Chen YR (1979) A new blind cobitid fish (Pisces, Cypriniformes) from subterranean waters in Yunnan, China. Dong Wu Xue Bao 25(3): 285–287. [In Chinese]
  • Chu XL, Chen YR (1990) The fishes of Yunnan, China. Part 2. Science Press, Beijing, 313 pp. [In Chinese]
  • Deng SQ, Wang XB, Zhang E (2022) Triplophysa qini, a new stygobitic species of loach (Teleostei: Nemacheilidae) from the upper Chang-Jiang Basin in Chongqing, Southwest China. Ichthyological Exploration of Freshwaters 1178: 1–11. https://doi.org/10.23788/IEF-1178
  • Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32(5): 1792–1797. https://doi.org/10.1093/nar/gkh340
  • Feng C, Zhou W, Tang Y, Gao Y, Chen J, Tong C, Liu S, Wanghe K, Zhao K (2019) Molecular systematics of the Triplophysa robusta (Cobitoidea) complex: extensive gene flow in a depauperate lineage. Molecular Phylogenetics and Evolution 132: 275–283. https://doi.org/10.1016/j.ympev.2018.12.009
  • He CL, Zhang E, Song ZB (2012) Triplophysa pseudostenura, a new nemacheiline loach (Cypriniformes: Balitoridae) from the Yalong River of China. Zootaxa 3586(1): 272–280. https://doi.org/10.11646/zootaxa.3586.1.26
  • Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh L (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution 35(2): 518–522. https://doi.org/10.1093/molbev/msx281
  • Huang TF, Zhang PL, Huang XL, Wu T, Gong XY, Zhang YX, Peng QZ, Liu ZX (2019) A new cave-dwelling blind loach, Triplophysa erythraea sp. nov. (Cypriniformes: Nemacheilidae), from Hunan Province, China. Zoological Research 40(4): 331–336. https://doi.org/10.24272/j.issn.2095-8137.2019.049
  • Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870–1874. https://doi.org/10.1093/molbev/msw054
  • Lan JH, Yang JX, Chen YR (1995) Two new species of the subfamily Nemacheilinae from Guangxi, China (Cypriniformes: Cobitidae). Dong Wu Fen Lei Xue Bao 20(3): 366–372. [In Chinese]
  • Lan JH, Gan X, Wu TJ, Yang J (2013) Cave Fishes of Guangxi, China. Science Press, Beijing. [In Chinese]
  • Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2017) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34(3): 772–773. https://doi.org/10.1093/molbev/msw260
  • Li WX (2004) The three new species of Cobitidae from Yunnan, China. Journal of Jishou University 25(3): 93–96. [Natural Science Edition] [In Chinese]
  • Li WX, Zhu ZG (2000) A new species of Triplophysa from cave Yunnan. Journal of Yunnan University 22(5): 396–398. [Natural Science Edition] [In Chinese]
  • Li WX, Yanf HF, Chen H, Tao CP, Qi SQ, Han NF (2008) A New Blind Underground Species of the Genus Triplophysa (Balitoridae) from Yunnan, China. Zoological Research 29(6): 674–678. https://doi.org/10.3724/SP.J.1141.2008.06674
  • Li J, Lan JH, Chen XY, Du LN (2017a) Description of Triplophysa luochengensis sp. nov. (Teleostei: Nemacheilidae) from a karst cave in Guangxi, China. Journal of Fish Biology 91(4): 1009–1017. https://doi.org/10.1111/jfb.13364
  • Li J, Li XH, Lan JH, Du LN (2017b) A new troglobitic loach Triplophysa tianlinensis (Teleostei: Nemacheilidae) from Guangxi, China. Ichthyological Research 64(3): [1–6]: 295–300. https://doi.org/10.1007/s10228-016-0565-0
  • Li XQ, Xiang XG, Jabbour F, Hagen O, Ortiz RDC, Soltis PS, Soltis DE, Wang W (2022) Biotic colonization of subtropical East Asian caves through time. Proceedings of the National Academy of Sciences 119(34): e2207199119. https://doi.org/10.1073/pnas.2207199119
  • Liu SW, Pan XF, Yang JX, Chen XY (2017) A new cave‐dwelling loach, Triplophysa xichouensis sp. nov. (Teleostei Nemacheilidae) from Yunnan, China. Journal of Fish Biology 90(3): 834–846. https://doi.org/10.1111/jfb.13201
  • Liu F, Zeng ZX, Gong Z (2022) Two new hypogean species of Triplophysa (Cypriniformes: Nemacheilidae) from the River Yangtze drainage in Guizhou, China. Journal of Vertebrate Biology 71(22062): 22062. https://doi.org/10.25225/jvb.22062
  • Lu ZM, Li XJ, Lü WJ, Huang JQ, Xu TK, Huang G, Qian FQ, Yang P, Chen SG, Mao WN, Zhao YH (2022) Triplophysa xuanweiensis sp. nov., a new blind loach species from a cave in China (Teleostei: Cypriniformes: Nemacheilidae). Zoological Research 43(2): 221–224. https://doi.org/10.24272/j.issn.2095-8137.2021.310
  • Luo T, Mao M-L, Lan C-T, Song L-X, Zhao X-R, Yu J, Wang X-L, Xiao N, Zhou J-J, Zhou J (2023) Four new hypogean species of the genus Triplophysa (Osteichthyes, Cypriniformes, Nemacheilidae) from Guizhou Province, Southwest China, based on molecular and morphological data. ZooKeys 1185: 43–81. https://doi.org/10.3897/zookeys.1185.105499
  • Nguyen LT, Schmidt HA, Von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
  • Ran JC, Li WX, Chen HM (2006) A new species of blind loach of Paracobitis from Guangxi, China (Cypriniformes: Cobitidae). Guangxi Shifan Daxue Xuebao. Ziran Kexue Ban 24(3): 81–82. [In Chinese]
  • Ren Q, Yang JX, Chen XY (2012) A new species of the genus Triplophysa (Cypriniformes: Nemacheilidae), Triplophysa longliensis sp. nov., from Guizhou, China. Zootaxa 3586(1): 187–194. https://doi.org/10.11646/zootaxa.3586.1.17
  • Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Tang L, Zhao YH, Zhang CG (2012) A new blind loach, Oreonectes elongatus sp. nov. (Cypriniformes: Balitoridae) from Guangxi, China. Environmental Biology of Fishes 93(4): 483–449. https://doi.org/10.1007/s10641-011-9943-7
  • Wang DZ, Li DJ (2001) Two new species of the genus Triplophysa from Guizhou, China (Cypriniformes: Cobitidae). Acta Zootaxonomica Sinica 16(1): 98–101. [In hinese]
  • Wen H, Luo T, Wang Y, Wang S, Liu T, Xiao N, Zhou J (2022) Molecular phylogeny and historical biogeography of the cave fish genus Sinocyclocheilus (Cypriniformes: Cyprinidae) in southwest China. Integrative Zoology 17(2): 311–325. https://doi.org/10.1111/1749-4877.12624
  • Wu TJ, Wei ML, Lan JH, Du LN (2018a) Triplophysa anshuiensis, a new species of blind loach from the Xijiang River, China (Teleostei, Nemacheilidae). ZooKeys 744: 67–77. https://doi.org/10.3897/zookeys.744.21742
  • Wu WJ, He AY, Yang JX, Du LN (2018b) Description of a new species of Triplophysa (Teleostei: Nemacheilidae) from Guizhou Province, China. Journal of Fish Biology 93(1): 88–94. https://doi.org/10.1111/jfb.13670
  • Xiao W, Zhang Y, Liu H (2001) Molecular systematics of Xenocyprinae (Teleostei: Cyprinidae): taxonomy, biogeography, and coevolution of a special group restricted in East Asia. Molecular Phylogenetics and Evolution 18(2): 163–173. https://doi.org/10.1006/mpev.2000.0879
  • Yang GY, Yuan FX, Liao YM (1986) A new blind Cobitidae fish from the subterranean water in Xiangxi, China. Huazhong Nongye Daxue Xuebao 5(3): 219–223. [In Chinese]
  • Yang JX, Chen XY, Lan JH (2004) Occurrence of two new Plateau indicator loaches of Nemacheilinae (Balitoridae) in Guangxi with Reference to Zoogeographical Significance. Zoological Research 25(2): 111–116. [In Chinese]
  • Yang J, Wu TJ, Yang JX (2012) A new cave-dwelling loach, Triplophysa macrocephala (Teleostei: Cypriniformes: Balitoridae), from Guangxi, China. Environmental Biology of Fishes 93(2): 169–175. https://doi.org/10.1007/s10641-011-9901-4
  • Yuan Z, Wu D, Wen Y, Xu W, Gao W, Dahn HA, Liu X, Jin J, Yu C, Xiao H, Che J. (2023) Historical mitochondrial genome introgression confounds species delimitation: evidence from phylogenetic inference in the Odorrana grahami species complex. Current Zoology 69(1): 82–90. https://doi.org/10.1093/cz/zoac010
  • Zhang CG, Shao GZ, Wu HL, Zhao YH (2020) Species Catalogue of China. Vol. 2. Animals, Vertebrates (V), Fishes. Science Press, Beijing. [In Chinese]
  • Zheng LP, Du LN, Chen XY, Yang JX (2009) A new species of Genus Triplophysa (Nemacheilinae: Balitoridae), Triplophysa longipectoralis sp. nov., from Guangxi, China. Environmental Biology of Fishes 85(3): 221–227. https://doi.org/10.1007/s10641-009-9485-4
  • Zhou QY, Chen PY (1993) Lithofacies change and palaeogeographical evolution during Late Cenozoic in Guizhou and its vicinity. Geology of Guizhou 10(3): 201–207.
  • Zhu SQ (1989) The loaches of the subfamily Nemacheilinae in China (Cypriniformes: Cobitidae). Jiangsu Science and Technology Publishing House, Nanjing, 150 pp. [In Chinese]
  • Zhu SQ, Cao WX (1988) Descriptions of two new species and a new subspecies of Noemacheilinae from Yunnan Province (Cypriniformes: Cobitidae). Dong Wu Fen Lei Xue Bao (1): 95–100. [In Chinese]

1Chang-Ting Lan, Li Wu, Tao Luo and Ye-Wei Liu contributed equally to this work.

Supplementary materials

Supplementary material 1 

Morphological characters and measurement data of the new species Triplophysa ziyunensis sp. nov., Triplophysa yaluwang sp. nov., T. wudangensis, T. rosa, T. qingzhenensis, and T. guizhouensis

Chang-Ting Lan, Li Wu, Tao Luo, Ye-Wei Liu, Jia-Jun Zhou, Jing Yu, Xin-Rui Zhao, Ning Xiao, Jiang Zhou

Data type: docx

Explanation note: *indicates the holotype.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (44.77 kb)
Supplementary material 2 

Results and percentage of variance explained by principal component analysis

Chang-Ting Lan, Li Wu, Tao Luo, Ye-Wei Liu, Jia-Jun Zhou, Jing Yu, Xin-Rui Zhao, Ning Xiao, Jiang Zhou

Data type: docx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (18.33 kb)
Supplementary material 3 

Specimens examined in this work

Chang-Ting Lan, Li Wu, Tao Luo, Ye-Wei Liu, Jia-Jun Zhou, Jing Yu, Xin-Rui Zhao, Ning Xiao, Jiang Zhou

Data type: docx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (23.64 kb)
login to comment