Received: 10 August 2019 Accepted: 4 November 2019 DOI: 10.1111/jfb.14191

REGULAR PAPER FISH

Oreonectes guilinensis (Teleostei, , ), a new loach species from Guangxi, China

Jin-Qing Huang1,2 | Jian Yang3 | Zhi-Qiang Wu1 | Ya-Hui Zhao2

1College of Life Science and Technology, Guangxi University, Nanning, China Abstract 2Key Laboratory of Zoological Systematics and A new loach Oreonectes guilinensis sp. nov. is described from Guangxi Zhuang Auton- Evolution, Institute of Zoology, Chinese omous Region, China. It is the second surface-dwelling fish identified in the genus Academy of Sciences, Beijing, China 3Key Laboratory of Environment Change and Oreonectes after the type species Oreonectes platycephalus. This new species can be Resources Use in Beibu Gulf, Nanning Normal distinguished from other species within the genus by the combination of the follow- University, Nanning, China ing features: a round caudal fin, 13–14 branched rays, an incomplete lateral line with Correspondence 4–6 pores, a short pelvic fin located some distance from the anus, a scale-covered Zhi-Qiang Wu, College of Life Science and – Technology, Guangxi University, Nanning, body, a stout build (body width 14.0% 16.7% of standard length), a posterior cham- 530004,China, ber of well-developed airbladder and a yellowish-brown body with a lateral dark Email: [email protected]; Ya-Hui Zhao, Key Laboratory of Zoological Systematics and brown strip. The maximum likelihood phylogenetic analysis based on cytb gene Evolution, Institute of Zoology, Chinese recovered this new species in a well-supported clade with the type species Academy of Sciences, Beijing, 100101, China, Email: [email protected] O. platycephalus, sister to the Lefua clade. Morphological comparisons as well as our phylogenetic tree support Oreonectes shuilongensis, Oreonectes daqikongensis, Funding information This study was supported by grants Oreonectes jiarongensis as species of Troglonectes. Based on our results there are (NSFC 31972868, 31860600, and 31970382) 6 valid species in the genus Oreonectes. from the National Nature Science Foundation of China, a grant (GEFC-15-16) from National KEYWORDS Geography Society, a grant (Y229YX5105) from the Key Laboratory of the Zoological Systematics new species, Oreonectes, Pearl River, phylogeny, surface-dwelling fish and Evolution of the Chinese Academy of Sciences, a grant from the Innovation Project of Guangxi Graduate Education (T3100098001), a grant from the Education Department of Guangxi Zhuang Automous Region, China and the Guangxi Natural Science Foundation (2017GXNSFFA198010).

1 | INTRODUCTION establishing a new genus of Nemacheilidae (i.e. Oreonectes Günther 1868). Oreonectes platycephalus has several diagnostic characteristics: Nemacheilidae is the second largest family of the order Cypriniformes a flat head, a slightly compressed body and a rounded caudal fin, and is ubiquitous in diverse freshwater environments in tropical Asia, dorsal-fin origin nearer to caudal-fin base than to snout tip and poste- Europe and north-east Africa. Nemacheilids are particularly abundant in rior to pelvic-fin base, the latter of which represents an important key swift-flowing hillside streams, where they are commonly found under characteristic of the genus Oreonectes (Günther, 1868). Over the next stones (Kottelat, 2012). China, one of the countries with the richest pop- 100 years, O. platycephalus was the only species identified in this ulation of nemacheilid loaches, has been reported to harbour loaches genus but Zheng (1981) described the second species of the genus belonging to 20 genera and more than 200 species (Zhang et al., 2016). Oreonectes based on seven specimens from a cave in Guangxi, China. Based on some specimens collected in Hong Kong, Günther Another cave species Oreonectes furcocaudalis was described by Zhu (1868) described a species he named Oreonectes platycephalus, and Cao (1987), who added another diagnostic characteristic for the genus Oreonectes: a short distance between the anterior and posterior urn:lsid:zoobank.org:pub:FC23EB97-C448-4351-8DE4-F2936D36C01A. urn:lsid:zoobank.org:act:638F8391-07D5-4A09-9B1A-65CC6ACED96D. nostril, with an anterior nostril in the form of a short tube extending

J Fish Biol. 2020;96:111–119. wileyonlinelibrary.com/journal/jfb © 2019 The Fisheries Society of the British Isles 111 112 FISH HUANG ET AL. into a relatively long barbel (Zhu, 1989). In the past 30 years, China. All activities followed Laboratory —Guideline for ethical equipped with these new diagnostic characters, dozens of cavefish review of animal welfare (GB/T 35892–2018). species were identified to belong to the genus Oreonectes. Specimens of the new species collected during field surveys in These cave-dwelling species of Oreonectes are still being discov- May 2016, in an open stream in Xingping, Guilin, Guangxi Zhuang ered and exhibit some substantial differences in terms of morphologi- Autonomous Region. Samples were collected using hand net and cal characteristics; i.e., shape of the caudal fin, position of the dorsal- mesh traps. Freshly caught fish were euthanised with eugenol. After fin origin and the caudal crests. While performing a review on the death, the pectoral fins from the right side were taken and preserved genus Oreonectes,Duet al. (2008) noticed this taxonomic problem in absolute alcohol for molecular analysis. Specimens used for mor- and tentatively divided the genus Oreonectes into two species groups; phological studies were preserved in 10% formalin, before being the platycephalus group with a rounded or truncated caudal fin, transferred to 70% ethanol for long-term storage at the collection of including Oreonectes anophthalmus Zheng 1981, O. platycephalus, the Institute of Zoology, Chinese Academy of Sciences, Beijing Oreonectes polystigmus Du, Chen & Yang 2008 and Oreonectes (ASIZB, institutional abbreviation is according to the list in Leviton retradorsalis Lan & Yang 1995 and the furcocaudalis group with a et al., 1985) and the Kunming Institute of Zoology, Chinese Academy forked caudal fin, including O. furcocaudalis and Oreonectes micro- of Sciences (KIZ). phthalmus Du, Chen & Yang 2008. Based on the original description The specimens examined (five species) for comparison belong to and drawings of Oreonectes, Kottelat (2012) suggested that some of ASIZB or KIZ. Methods used for counting and measurements were the previously identified species were not species of Oreonectes due according to Tang et al. (2012) and characteristics of cephalic lateral to their forked (v. rounded) caudal fin and their dorsal-fin originating line system were examined following guidelines from Kottelat (1990). clearly in front of the pelvic-fin origin (v. conspicuously behind the All measurements were taken point to point with dial callipers to the pelvic-fin base). These suggested species were Oreonectes elongatus nearest 0.1 mm. A few of the finer characteristics (i.e., cephalic lateral- Tang, Zhao & Zhang 2012, O. furcocaudalis, Oreonectes macrolepis Huang, line system, fins rays, nostrils distance) were examined under a micro- Du, Chen & Yang 2009, O. microphthalmus and Oreonectes translucens scope. Vertebral counts including the Weberian apparatus and the last Zhang, Zhao & Zhang 2006. Tang (2012) also came across a similar con- half-centrum were examined by micro-computed tomography (m-CT) clusion on the species within Oreonectes and suggested that a new genus images (Xradia MicroXCT-400, 30 V; Zeiss; www.zeiss.com). should be established for these species, which all possess a forked caudal A fragment of the mitochondrial gene coding for cytochrome b Š fin, a dorsal-fin origin in front of the pelvic-fin origin and well-developed (cytb) was amplified with the primers designed by lechtová et al. μ μ μ caudal crests. Subsequently, Zhang, Zhao and Tang (2016) established (2006). The 50 l PCR reactions included 5 l PCR buffer (x 10), 5 l μ μ the genus Troglonectes Zhang, Zhao & Tang 2016 with O. furcocaudalis of 2 mM deoxynucleotide triphosphate (dNTP), 2.5 lof2 M primers, 0.5 μl Taq DNA polymerase, 1.0 μl of extracted DNA and as type species, including those species with forked caudal fins and cau- 36 μlofHO. The PCR profile was 5 min at 94C, followed by dal crests, which were identified as distinct from Oreonectes (Zhang et al., 2 35 cycles of 45 s at 94C, 30 s at 54C, 1 min at 72C and ending with 2016). Meanwhile, Zhang et al. (2016) revised the genus Oreonectes as a final extension of 7 min at 72C. Amplification was visually checked having the following major diagnostic characteristics: a depressed head, on 1.0% agarose gels and PCR products were purified using a univer- nostrils separated by a short distance, anterior nostril in the form of a sal DNA clean-up kit. We obtained cytb sequences of another 23 spe- short tube that extends into a short barbel, a dorsal fin located far behind cies in the genera Oreonectes, Troglonectes, Triplophysa Rendahl 1933, the origin of the ventral and a rounded or truncated caudal fin. Currently McClelland 1838, Lefua Herzenstein 1888 and Nemacheilus there are 8 valid species in the genus Oreonectes,mostofwhichare Bleeker 1863 from GenBank (Table 1). A maximum likelihood cavefishes with narrow distributions, the only exception being the type (ML) tree building method was used to reconstruct the phylogenetic species O. platycephalus, which is the only surface-dwelling fish that is tree using MEGA 7.0, while reliability was tested using the general broadly distributed in the south of China (Zhu, 1989; Du et al., 2008) and time reversible model and bootstrap method by repeating 1000 times. North Vietnam (Kottelat, 2001). In 2016, 8 specimens were collected from an open stream in Xingping, Yangshuo County, Guilin City, Guangxi Zhuang Autonomous 3 | RESULTS Region, China. Examining these specimens, we observed several dis- tinctive characteristics in these specimens compared with other con- 3.1 | Oreonectes guilinensis sp. nov. (Figures 1, 2 and generic species, prompting us to describe them as a new species of Table 2) the genus Oreonectes.

3.1.1 | Holotype 2 | MATERIALS AND METHODS

ASIZB208002, 66.3 mm standard length (LS), from Xingping, Yangshuo Field collections followed the Guide to Collection, Preservation, Iden- County, Guilin City, Guangxi Zhuang Autonomous Region, China, a tification and Information Share of Animal Specimens (Xue, 2010) and tributary of Lijiang river, Pearl River system (24 530 30.7700 N, 110 Implementation rules of Fisheries Law of the People's Republic of 330 45.6200 E), collected in May 2016 by Jian Yang. HUANG ET AL. FISH 113

TABLE 1 GenBank accession numbers for the samples of loach from southern China included in the phylogenetic analysis

Genus Species Accession number

Oreonectes guilinensis sp.nov. MN239094 platycephalus DQ105197 jiarongensis KU987437 shuilongensis KF640641 daqikongensis KU987436 Troglonectes furcocaudalis NC032384 Triplophysa xiangxiensis JN696407 stewarti DQ105248 rosa NC019587 bleekeri FJ406605 orientalis DQ105251 wuweiensis KX373838

tenuis KX373841 FIGURE 1 Oreonectes guilinensis sp. nov. Holotype ASIZB Schistura fasciolata HM010565 208002, standard length 66.3 mm preserved specimens caudofurca JN837651 desmotes GQ174368 callichroma JN837652 latifasciata JN837653 bucculenta JN837654 Lefua costata DQ105196 nikkonis AB100919 echigonia AB080180 Nemacheilus masyae GQ174377 ornatus GQ174363 pallidus GQ174370 FIGURE 2 Live Oreonectes guilinensis sp. nov 3.1.2 | Paratypes postocular head length. Mouth inferior, curved. Upper and lower ASIZB208001, ASIZB208003-208007, KIZ2016007378, lips smooth. Three pairs of barbels: inner rostral extending anterior 51.9–73.0 mm LS; data as for holotype. margin of eye, outer rostral and maxillary barbels extending over posterior margin of eye. Nostrils separate with a short distance. 3.1.3 | Diagnosis Anterior nostril in a short tube, which extend into a short barbel.

The new species is distinguished from other species of the genus Eyes normal. – Oreonectes. by the combination of following characteristics: caudal fin Dorsal-fin rays ii-6, pectoral-fin rays i-9-10, pelvic-fin rays i 6, – – rounded; pelvic fin short, with a short distance to anus, body (except anal-fin rays iii 5, caudal fin branched 13 14. Dorsal fin short, origin head) completely covered by scales; lateral line incomplete, with 4–6 closer to caudal-fin base than to snout tip, posterior to pelvic-fin ori- gin, distal margin round. Pectoral fin almost reaching half distance to pores; body width (WB) 14.8%–16.4% LS (v. 8.8%–12.3% in conge- ners); body yellowish-brown, with a dark brown lateral stripe on the pelvic fin. Pelvic fin with a short distance (2.5 to 4 mm) to anus. Anal flank. Two chambers of air-bladder, posterior chamber developed, fill- fin origin next to anus with a short distance. Caudal fin rounded. Cau- ing body cavity, connected with anterior chamber by a long, dal peduncle without adipose crests along both dorsal and ventral slender tube. sides. Vertebra 4 + 33–36. Except head, body completely covered by scales; lateral line incomplete, with 4–6 pores; cephalic lateral-line system, with 6–9 3.1.4 | Description supraorbital, 4 + 5–9 infraorbital pores, 3–4 canal pores, with 3–7 Body elongate and cylindrical, with depth decreasing from dorsal- preoperculo-mandibular canal pores. Two chambers of air-bladder, pos- fin origin to caudal-fin base. Head slightly depressed and flattened, terior chamber developed, filling body cavity, connected with anterior width greater than depth. Snout round, snout length shorter than chamber by long, slender tube (Figure 3). TABLE 2 Meristics and morphometrics of six species from the genus Oreonectes 114

Character O. guilinensis sp. nov. O. platycephalus O. anopthalmus O. guananensis O. polystigmus O. luochengensis

Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD

No. of specimen 8 10 3 6 11 5 FISH Dorsal fin 7 7 7 7 7 7 Anal fin 5 7 5 5 5 5 Pectoral fin 9–10 9–10 10 11–12 10–11 11–12 Pelvic fin 6–77 4 7–86–77 Caudal fin 13–14 14–16 12 15–16 14–15 15–17 Lateral line pores 4–613–17 – 7–10 5–14 11–13

Standard length (LS, mm) 51.9–73.0 65.0 ± 6.1 46.2–72.5 52.3 ± 7.5 26.5–40.1 34.8 ± 7.3 53.0–76.6 63.9 ± 9.9 34.3–58.6 45.4 ± 8.3 66.8–73.9 71.6 ± 2.9

% LS Body height 16.5–18.7 17.5 ± 0.8 11.5–15.8 13.6 ± 1.5 11.6–13.6 12.6 ± 1.0 15.1–18.3 16.5 ± 1.1 11.0–16.7 14.3 ± 1.7 14.6–17.2 16.3 ± 1.0 Maximum body height 17.–20.0 18.7 ± 0.8 12.3–17.1 14.8 ± 1.7 12.8–14.2 13.4 ± 0.7 15.8–19.3 17.5 ± 1.2 13.9–19.4 16.3 ± 1.9 16.4–17.8 17.2 ± 0.5 Body width 14.0–16.7 15.5 ± 0.9 8.3–9.4 8.8 ± 0.3 9.6–11.8 10.5 ± 1.2 11.3–13.1 12.3 ± 0.6 6.5–12.6 8.5 ± 1.9 12.1–12.5 12.3 ± 0.2 Maximum body width 17.2–19.8 18.4 ± 0.9 10.6–15.4 13.0 ± 1.4 13.2–15.9 14.3 ± 1.4 13.9–16.8 15.0 ± 1.0 11.5–17.1 13.8 ± 1.7 14.8–16.0 15.4 ± 0.5 Body width at anus 11.5–13.5 12.4 ± 0.6 7.2–9.1 8.3 ± 0.6 7.9–10.3 9.2 ± 1.2 9.0–10.4 9.7 ± 0.5 5.3–10.1 7.0 ± 1.6 9.6–10.0 9.8 ± 0.2 Predorsal length 57.0–61.6 59.6 ± 0.6 57.3–61.0 59.4 ± 1.2 60.5–64.5 62.4 ± 2.0 56.7–58.0 57.3 ± 0.5 55.8–63.3 60.2 ± 2.3 56.0–58.0 58.3 ± 0.8 Dorsal-fin length 15.9–19.1 17.5 ± 1.0 17.6–19.9 19.1 ± 0.7 13.7–17.0 15.4 ± 1.6 15.7–18.7 17.1 ± 0.9 16.3–20.6 18.1 ± 1.2 17.7–19.1 18.6 ± 0.5 Dorsal-fin base length 6.6–10.2 9.0 ± 1.1 8.7–11.5 10.0 ± 0.7 9.4–10.8 9.9 ± 0.7 9.1–9.6 9.4 ± 0.2 8.6–12.1 10.2 ± 1.2 10.1–10.5 10.3 ± 0.2 Prepelvic length 54.7–59.0 56.5 ± 1.7 48.1–51.3 49.8 ± 1.0 58.5–61.3 59.6 ± 1.5 53.2–55.4 54.0 ± 0.8 50.4–55.6 53.6 ± 1.7 51.9–57.9 54.0 ± 2.4 Pelvic-fin length 12.0–13.9 13.2 ± 0.6 16.9–19.1 18.1 ± 0.7 9.9–10.8 10.3 ± 0.5 12.2–13.7 13.1 ± 0.5 12.9–15.9 13.9 ± 1.0 12.2–13.2 12.5 ± 0.4 Pelvic-fin base length 3.1–3.4 3.3 ± 0.1 3.5–5.7 4.2 ± 0.7 1.9–2.9 2.5 ± 0.5 3.3–3.7 3.5 ± 0.2 2.3–3.6 2.9 ± 0.5 3.0–3.7 3.3 ± 0.2 Pre-anus length 71.4–75.9 73.3 ± 1.5 63.8–70.5 67.8 ± 2.0 68.6–72.2 69.9 ± 2.0 68.9–71.8 70.6 ± 1.0 67.3–73.2 71.1 ± 1.6 68.4–73.5 70.9 ± 1.8 Pre-anal length 77.2–82.9 80.5 ± 1.9 72.7–79.0 75.7 ± 2.1 74.3–77.4 75.4 ± 1.7 755.9–78.1 77.0 ± 0.9 73.0–78.5 77.1 ± 1.7 75.0–79.4 77.8 ± 1.7 Anal fin length 14.0–16.0 15.0 ± 0.7 16.8–20.6 18.4 ± 1.3 11.7–15.3 13.4 ± 1.8 13.7–15.4 14.3 ± 0.8 14.3–19.9 15.8 ± 1.5 15.1–16.4 15.8 ± 0.5 Anal-fin base length 6.0–7.2 6.7 ± 0.4 7.8–10.7 8.9 ± 0.9 5.9–9.4 7.9 ± 1.8 6.8–8.4 7.4 ± 0.6 2.5–9.7 8.0 ± 2.0 7.3–7.7 7.4 ± 0.2 Prepectoral length 20.8–24.8 23.5 ± 1.3 19.6–23.4 21.6 ± 1.0 24.0–26.1 25.4 ± 1.2 20.3–22.6 21.0 ± 0.9 19.9–25.5 23.1 ± 1.8 19.2–20.9 19.8 ± 0.6 Pectoral-fin length 15.6–17.5 16.6 ± 0.7 16.8–20.4 18.7 ± 1.1 13.1–15.2 13.9 ± 1.1 15.5–16.6 16.1 ± 0.4 13.4–18.6 15.3 ± 1.7 15.8–17.2 16.6 ± 0.6 Pectoral-fin base length 3.1–4.5 3.7 ± 0.5 3.5–5.4 4.7 ± 0.6 3.6–5.0 4.3 ± 0.7 3.5–4.8 4.0 ± 0.5 2.8–5.1 3.5 ± 0.6 3.9–4.5 4.4 ± 0.3 Caudal-fin length 19.8–23.1 21.5 20.5–24.7 20.7 ± 1.5 13.6–19.1 15.9 ± 2.9 18.4–20.3 19.1 ± 0.8 13.5–23.1 19.1 ± 2.5 16.8–18.2 17.5 ± 0.5 Caudal-peduncle length 11.9–18.7 15.8 14.9–18.9 16.1 ± 1.1 17.3–18.2 17.7 ± 0.5 15.0–16.3 15.4 ± 0.5 14.2–16.5 15.2 ± 0.8 14.0–16.3 15.8 ± 0.8

Caudal-peduncle depth 10.5–11.7 11.1 8.0–12.1 10.8 ± 1.1 8.9–10.2 9.7 ± 0.6 9.4–10.9 10.3 ± 0.5 7.3–11.5 8.5 ± 1.4 9.2–10.3 9.8 ± 0.5 HUANG

Head length (LH) 19.9–23.3 21.4 20.4–21.7 21.0 ± 0.4 24.3–25.4 24.9 ± 0.5 19.8–20.7 20.0 ± 0.3 16.6–24.9 22.4 ± 2.2 19.6–20.5 20.0 ± 0.4

(Continues) AL ET . HUANG TAL ET .

TABLE 2 (Continued)

Character O. guilinensis sp. nov. O. platycephalus O. anopthalmus O. guananensis O. polystigmus O. luochengensis

Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD

% LH Head height at eye 44.4–50.3 46.9 ± 2.2 39.1–46.2 42.2 ± 2.3 –– 50.1–54.9 52 ± 2.0 37.9–47.4 41.7 ± 2.9 46.0–50.2 48.3 ± 1.8 Head height at nape 54.5–63.0 57.8 ± 3.6 45.2–53.3 49.8 ± 3.1 47.8–59.2 52.1 ± 6.2 59.7–64.4 61.8 ± 1.8 47.0–55.4 50.9 ± 3.2 56.2–68.1 61.0 ± 4.7 Head width at eye 57.3–71.3 64.2 ± 4.4 50.8–64.0 57.9 ± 3.9 –– 66.7–71.1 69 ± 1.5 17.0–60.2 45.8 ± 11.4 59.472.8 65.1 ± 5.0 Maximum head width 76.5–83.6 79.7 ± 3.0 63.8–72.3 68.4 ± 3.2 62.3–77.7 70.6 ± 7.8 77.5–83.8 80.7 ± 2.0 57.2–69.9 63.0 ± 4.2 66.3–85.9 73.4 ± 7.6 Snout length 37.1–42.3 40.8 ± 1.7 37.8–45.3 41.7 ± 2.2 –– 32.1–39.6 35.2 ± 2.5 32.6–40.9 36.9 ± 2.9 34.0–41.2 36.1 ± 2.9 Outer rostral barbel length 57.9–70.0 63.5 ± 4.0 47.655.2 52.3 ± 2.3 23.6–39.4 33.3 ± 8.5 61.8–78.6 72.9 ± 6.2 43.0–73.1 58.2 ± 8.0 47.8–63.6 54.7 ± 7.0 Maxillary barbel length 49.8–66.8 57.6 ± 4.9 43.7–48.8 45.8 ± 1.7 24.6–36.2 28.6 ± 6.6 58.0–71.2 62.6 ± 4.9 41.8–62.7 49.7 ± 6.2 45.2–60.2 50.7 ± 6.7 Postocular head length 43.5–52.5 48.8 ± 3.1 48.0–56.4 51.1 ± 2.8 –– 44.3–51.2 47.6 ± 2.8 47.3–53.7 50.6 ± 1.9 44.6–52.7 47.1 ± 3.2 Eye diameter 9.7–14.1 11.6 ± 1.5 14.5–18.5 16.3 ± 1.6 –– 13.4–15.9 14.6 ± 0.9 13.2–17.5 15.5 ± 1.6 11.4–12.5 12.0 ± 0.45 Interorbital width 40.5–44.3 42.1 ± 1.3 39.9–46.2 43.5 ± 2.4 –– 45.8–50.1 48.0 ± 1.9 35.7–46.1 38.7 ± 3.6 39.0–41.2 40.2 ± 1.0 Length of anterior nostril tube 8.5–13.0 10.9 ± 1.8 13.4–19.0 16.2 ± 1.8 5.0–6.6 5.9 ± 0.8 11.8–15.2 13.3 ± 1.3 7.7–12.8 9.5 ± 1.7 12.3–14.3 13.3 ± 0.9 and barbel Pre-anterior nostril length 19.7–26.3 22.1 ± 2.3 15.0–21.1 18.3 ± 2.0 14.1–18.4 15.8 ± 2.2 17.0–19.0 18.1 ± 0.7 12.9–19.1 16.7 ± 2.0 18.8–19.7 19.2 ± 0.4 FISH Distance between anterior and 5.7–9.1 7.4 ± 1.2 4.7–77 6.2 ± 0.9 3.4–4.1 3.6 ± 0.4 3. 4–4.9 4.5 ± 0.6 5.3–7.8 6.5 ± 0.7 5.5–8.2 6.3 ± 1.1 posterior nostrils Distance between posterior 31.3–37.6 34.4 ± 2.0 36.–42.8 40.0 ± 1.8 38.2–41.2 39.8 ± 1.5 35.3–39.1 37.2 ± 15 31.2–37.8 34.8 ± 2.0 31.4–34.1 32.9 ± 1.0 nostrils Distance between eye and 4.8–7.2 6.0 ± 1.0 3.0–4.5 3.8 ± 0.5 –– 1.7–4.4 3.1 ± 0.9 3.1–5.7 4.0 ± 0.8 3.3–4.0 3.7 ± 0.2 posterior nostril 115 116 FISH HUANG ET AL.

3.1.5 | Coloration colour, except body a uniform dull yellow, turn into pale brown over time (Figure 1). Body yellowish-brown with a dark brown lateral stripe on flank, many irregularly shaped speckles, anal-fin hyaline, other fins with irregularly black dots (Figure 2). Colour in alcohol similar to life 3.1.6 | Distribution

This new species is only found Shigumen village Xingping town Yangshuo County, Guilin City, Guangxi Zhuang Autonomous Region of southern China (24 530 30.7700 N, 110 330 45.6200 E; Figure 4).

3.1.7 | Etymology

The species name derives from the Chinese name Guilin, the name of the city where the specimens were collected.

3.1.8 | Genetic comparisons

The molecular phylogenies from the ML analyses (Figure 5) show that Oreonectes guilinensis sp. nov. and the type species O. platycephalus form a monophyletic group with strong support (100 bootstrap), sister to the Lefua clade. Interestingly, these controversial species including Oreonectes shuilongensis Deng, Xiao, Hou & Zhou 2016, Oreonectes daqikongensis Deng, Wen, Xiao & Zhou 2016 and Oreonectes jiarongensis (Lin, Li & Song 2012) were resolved in a well-supported clade with Troglonectes furcocaudalis (Zhu & Cao 1987) (100 boot- FIGURE 3 Air-bladder of Oreonectes guilinensis sp. nov. (a), strap). On this basis, as well as morphological comparisons, we are Anterior chamber; (b), posterior chamber; (c), a long slender tube confident in assigning the new species to the genus Oreonectes.

Guizhou China

N

Guilin City

Lijiang River

Liujiang River Guangxi

Guangdong Hongshuihe River

040 Xunjiang River Xijiang River km

FIGURE 4 Location ( ) in southern China where Oreonectes guilinensis sp. nov. was found HUANG ET AL. FISH 117

100 Oreonectes guilinensis characteristics of Troglonectes: a forked caudal fin, a dorsal fin origi- Oreonectes platycephalus 59 nating opposite to the pelvic-fin origin and developed caudal crests. Lefua echigonia Liu et al. (2019) considered O. shuilongensis as a species of Oreonectes Lefua costata 77 99 100 Lefua nikkonis and it was embedded within T. furcocaudalis, O. daqikongensis and Troglonectes furcocaudalis O. jiarongensis in phylogenetic tree, which did not support the results Troglonectes daqikongensis 52 99 of Zhang et al. (2016). However, according to morphological compari- 39 Troglonectes shuilongensis 37 Troglonectes jiarongensis sons, as well as our phylogenetic-tree supports, the result of Zhang Schistura lafasciata et al. (2016) and O. shuilongensis, O. daqikongensis and O. jiarongensis Schistura bucculenta 100 as species of Troglonectes (Figure 5). Both morphological and molecu- Schistura fasciolata 28 lar data revealed that O. guilinensis is a new species of Oreonectes.We 34 Schistura caudofurca 100 Schistura callichromus found that only O. platycephalus, O. polystigmus, O. guananensis, Nemacheilus ornatus Oreonectes luochengensis Yang, Wu, Wei & Yang 2011, Oreonectes 100 Nemacheilus pallidus 100 Nemacheilus masyai anophthalmus and O. guilinensis sp. nov. shared the common charac-

100 Triplophysa xiangxiensis teristics of the genus Oreonectes and considered them to be the valid Triplophysa rosa species in the genus Oreonectes. 53 Triplophysa bleekeri 96 Triplophysa stewar Oreonectes guilinensis sp. nov. and O. anophthalmus exhibit a

100 Triplophysa wuweiensis rounded caudal fin and a dorsal-fin originating posterior to the pelvic- 93 Triplophysa orientalis fin origin. However, this new species can be very easily distinguished 76 Triplophysa tenuis from O. anophthalmus due to its normal eyes, pigmentation, scale-

0.05 covered body (v. eyeless, body pigmentation degradation, scale-less body), 6 pelvic-fin rays (v. 4) and the absence of crests (v. well- FIGURE 5 Maximum likelihood phylogenetic tree of species developed). within the family Nemacheilidae Although O. luochengensis is one of the cavefish species in this genus (Yang et al. 2011a) and shares the same characteristics of the dorsal fin, eyes and incomplete lateral line with the new spices, 4 | DISCUSSION O. guilinensis sp. nov. can be very easily distinguished from it by the presence of pigmentations on the body and fins (v. without any pig-

Although Zhang et al. (2016) revised the genus Oreonectes and mentation on the body and fins), 4–6 lateral line pores (v.11–13), WB – – established the genus Troglonectes based on a forked caudal fin, a dor- 14.0% 16.7% LS (v. 12.1% 12.5%), a rounded caudal fin (v. truncated) sal fin originating opposite to or slightly in front of the pelvic-fin origin and 13–14 caudal-fin rays (v.15–17). and developed caudal crests; there remains some species with forked Oreonectes guilinensis sp. nov. and O. platycephalus are the only caudal fins that were not assigned to Oreonectes (Deng et al. 2016a, known surface species in the genus. Both share a similar body shape Deng et al. 2016b, Liu et al. 2019). Although Deng et al. (2016a) and colour pattern, normal eyes, a rounded caudal fin and a dorsal-fin originating posterior to the pelvic-fin origin. However, the new species described a specimen as O. shuilongensis from Guizhou, China, we ver- differs from O. platycephalus by its developed posterior chamber of air- ified the specimens labelled as Oreonectes shuilongensis from the type bladder (v. degraded), its pelvic-fin tip within a short distance to the locality with the original description and found these do not belong to anus (v. reaching to the anus) when adpressed backwards, having 4–6 the genus Oreonectes due to their slightly concave caudal fin. In the lateral line pores (v.13–17) and W 14.0%–16.7% L (v.8.3%–9.5%). same year, Deng et al. (2016b) described another species, Oreonectes B S Oreonectes guilinensis sp.nov.andO. guananensis (Yang et al. daqikongensis, from Guizhou, China. Again, we examined the 2011b) share the following characteristics: a normal developed topotypes based on the original description and found that this spe- eye, a pigmented body, a dark brown lateral stripe on the body and cies belongs to the genus Troglonectes due to the forked caudal fin, a two chambers of the air-bladder, with a developed posterior cham- dorsal fin originating slightly in front of the pelvic-fin origin and devel- ber. However, this new species differs from O. guananensis by hav- oped caudal crests. Therefore, O. daqikongensis should be classified as ing 13–14 caudal-fin rays (v.15–16), 9–10 pectoral-fin rays Troglonectes daqikongensis. Based on morphological characteristics (v.11–12), pectoral fins, dorsal-fin rays and caudal fin with black and molecular data, Liu et al. (2019) reassigned Triplophysa jiarongensis spots (v. all fins hyaline) and WB 14.0%–16.7% LS (v. to Oreonectes. Due to the anterior and posterior nostrils of 11.3%–13.1%). Triplophysa jiarongensis were separated by a short distance and there The closest known relative species to the new species described was no secondary sexual dimorphism in male specimens, which were here appears to be O. polystigmus. Both of them follow a similar pat- not fit the diagnostic characteristics of Triplophysa (Liu et al., 2019). tern of distribution in Guilin, Guangxi. They also share the following Although we agreed that Triplophysa jiarongensis is not a species of characteristics: a rounded caudal fin, a dorsal-fin origin vertically Triplophysa, we do not believe that it should be classified as opposite pelvic-fin root, pelvic-fin tip within a short distance to the Oreonectes, since examining the topotypes showed diagnostic anus when adpressed backwards, incomplete lateral line and a 118 FISH HUANG ET AL. developed posterior chamber of the air-bladder. However, 5.5 | Oreonectes anophthalmus O. guilinensis sp. nov. and O. polystigmus differ in the maxillary barbel ASIZB60294 (holotype), ASIZB60287, ASIZB70022 26.5–40.3 mm L . reaching the posterior margin of the eye (v. reaching the vertical of S All 3 specimens checked here are from Qifeng mountain, Xiahuan vil- the pectoral-fin origin) and WB 14.0%–16.7% LS (v. 6.5%–12.6%). lage, Wuming, Guangxi, China; Nov.1977.

4.1 | Key to species of Oreonectes ACKNOWLEDGEMENTS 1. Eyes absent, branched pelvic-fin rays 4...... O. anophthalmus We are grateful to E. Lunghi for proofreading this manuscript. We Eyes present, branched pelvic-fin rays 6–7...... 2 express particular thanks to L. Tang and X. J. Song for checking the 2. Body pigment absent, caudal fin truncated...... O. luochengensis holotype, paratypes and species of O. polystigmus, O. guananensis and 3. Body with pigments, caudal fin rounded...... 4 O. luochengensis. We thank X. J. Li for the photograph of the holotype. 4. Posterior chamber of air-bladder degenerated, dorsal fin placed far backwards, at some distance behind the root of ven- trals...... O. platycephalus AUTHOR CONTRIBUTIONS Posterior chamber of air-bladder developed, dorsal-fin origin verti- J. Q. H., Z. Q. W. and Y. H. Z. designed the study. J. Y. contributed to cal to the root of pelvic fin...... 5 the field work. J. Q.H. collected data, performed data analyses and 5. Body without obvious speckles, hyaline on all wrote the manuscript with input from all authors. J. Q. H., J. Y. and fins...... O. guananensis Y. H. Z. revised the manuscript. All authors read and approved the Body with many obviously speckles, dorsal fin and caudal fin with final version of the manuscript. irregular black strips...... 6

6. Maxillary barbel reaching the vertical of the pectoral-fin origin, WB – 6.5% 12.6% LS ...... O. polystigmus ORCID Maxillary barbel not reaching the vertical of the pectoral-fin origin, Ya-Hui Zhao https://orcid.org/0000-0002-4615-596X WB 14.0%–16.7% LS...... O. guilinensis sp. nov.

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