﻿A new species of Homatula (Teleostei, Cobitoidea, Nemacheilidae) from the Pearl River drainage, Yunnan, China

﻿Abstract Based on morphological and molecular analysis of Homatula species distributed in the Nanpanjiang River in Yunnan, China, we described a new species, Homatularobustasp. nov. It differs from its congeners by a combination of the following characters: naked and robust body with well-developed crests (caudal peduncle depth as a percentage of its length: 70.5–78.5%); lateral line complete; median notch on lower jaw; median gap on lower lip; three pairs of short barbels, with maxillary barbels extending posteriorly to anterior edge of eyes; branched dorsal-fin rays 8½; and vertebrae 37–39. It can further be distinguished from H.nanpanjiangensis by several differences of the caudal skeleton such as the number of hypural elements, the presence of epurale and the shape of neural and haemal spines. Phylogenetic analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene indicated that the new species represents an independent lineage. It is separated from other Homatula species by a minimum of 5.3% Kimura-2-parameter distance in the COI gene. Furthermore, we confirmed that Homatulawenshanensis should be a member of Homatula based on both skeleton and molecular evidence.


Introduction
Homatula, a group of benthic nemacheilids distributed in the eastern slope of the Qinghai-Tibetan Plateau, was established by Nichols in 1925 based on Nemachilus potanini Günther, 1896 from the Minjiang River (a tributary of the Yangtze River, Sichuan, China) (Kottelat 1990;Bănărescu and Nalbant 1995;Min et al. 2012). Species of Homatula are characterized by the crests along the dorsal and ventral margins of the caudal peduncle supported by rudimentary procurrent caudalfin rays, the presence of a degenerated non-ossified secondary gas-bladder chamber, and a medium to large-sized body with a maximum standard length of 190 mm (Zhu 1989;Kottelat 1990;Bănărescu and Nalbant 1995;Endruweit et al. 2018).
Currently, 21 valid species are recognized, mostly distributed in China, and only one species is recently reported from Vietnam (Nguyen et al. 2021;Zhou et al. 2021). In China, six species are in the Palaearctic drainages of the Yangtze and Yellow River, four in the Lancangjiang River (upper reaches of the Mekong River), three in the Nujiang River (Salween River), four in the Red River, and three in the Nanpanjiang River (upper reaches of the Pearl River), respectively. None of them is distributed across these large river systems.
Three Homatula species have been reported from the Nanpanjiang River, i.e., H. oligolepis (Cao & Zhu, 1989), H. longidorsalis (Yang, Chen & Kottelat, 1994) and H. nanpanjiangensis (Min, Chen & Yang, 2010). In 2009, a medium-sized loach was collected from Luoping County, Yunnan Province, China, which belongs to the Nanpanjiang River drainage. By comparing it to other Homatula species, especially the species distributed in the Nanpanjiang River, we describe it as a new species here.

Materials and methods
All specimens were collected in 2009 from Yunnan Province, China and they were fixed either in 95% ethanol or 10% formalin and transferred to 75% ethanol for preservation. For DNA analysis, tissue samples from the left pelvic fin were excised from one or more specimens and placed in 95% ethanol. General methods for measurements and counts were done following Kottelat (1990), pore counts followed Armbruster (2012). Measurements were made with digital calipers to the nearest 0.1 mm from the left side. X-ray images were used to count vertebrae and simple fin rays. Lateral line pores and rays of paired fins were counted under a binocular microscope. The Weberian apparatus was counted as four vertebrae. Caudal vertebrae encompassed all centra bearing a haemal spine, including the urostyle, which was counted as one vertebra. Eye diameter was measured horizontally. Body depth was measured at the dorsal-fin origin. Lateral head length was measured from snout tip to the posterior margin of the operculum, excluding the opercular membrane. Examined specimens were deposited in the collection of the Kunming Natural His-tory Museum of Zoology, Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences.
In order to compare skeletal morphology, we applied Computed Microtomographic (μCT) scans of the holotype of H. robusta (KIZ 2009000125), a paratype of H. nanpanjiangensis (KIZ 1994000029) and a specimen of H. wenshanensis (KIZ 2014005686). Specimens were scanned using a GE Phoenix v|tome|x m dual tube 300/180 kV system in the Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Chinese Academy of Sciences. The specimen was scanned with an energy beam of 80 kV and a flux of 80*μA using a 360° rotation and then reconstructed into a 4096*4096 matrix of 1536 slices. The final CT reconstructed skeleton images were exported with a minimum resolution of 6.099 μm. The skeleton images were exported from the virtual 3D model which was reconstructed using Volume Graphics Studio 3.0. Osteological terminology generally follows that of Prokofiev (2009) andConway (2011) with modifications.
DNA was extracted from fin tissues using standard phenol-chloroform extraction (Sambrook et al. 1989). Mitochondrial cytochrome c oxidase subunit 1 (COI) was amplified by polymerase chain reaction (PCR). The PCR protocols were conducted in 50-μl reactions as follows: initial denaturation step at 95 °C for 5 min, 35 cycles at 94 °C for 30 s, 56 °C for 45 s, and 72 °C for 1 min, and final extension at 72 °C for 10 min. The primers used for COI were LCOIa (CCT ACC TgT ggC AAT CAC RCg C), HCOI (gTg AAT Agg ggg AAT CAg Tg) . Fragments were sequenced by the Shanghai DNA Biotechnologies Company (China). DNA sequences were aligned using default settings in MAFFT v7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013), and, if necessary, adjusted by eye. MEGA7 (Kumar et al. 2016) was used to calculate the Kimura's 2-parameter genetic distance (K2P). The phylogeny was analyzed using MrBayes 3.2 (Ronquist et al. 2012) with the generalized time reversible model (nst = 6) and the gamma-distributed rate variation and proportion of invariable positions (GTR+ I) for the COI datasets. We ran four simultaneous Monte Carlo Markov chains for 2 000 000 generations, with sampling every 1000 generations, and the first 25% of samples were discarded as burn-in.
Snout blunt in lateral view, cheeks inflated. Eyes elliptical horizontally, dorsolaterally positioned. Mouth inferior, slightly arched. Anterior nostril in flap, next to posterior nostril. Lips moderately thick, upper lip smooth, slightly notched medially, lower lip with shallow furrows and median gap. Processus dentiformis on upper jaw present with circular arc edge; lower jaw spoon-like with a median notch. Three pairs of barbels, maxillary barbel reaching anterior margin of eye, outer rostral barbel reaching inner corner of mouth and inner rostral barbel not.
Dorsal-fin rays iv, 8½, distal margin slightly convex. Pectoral-fin rays 11, reaching about halfway from insertion of pectoral fin to insertion of pelvic fin. Pelvic-fin rays 6-8, reaching close to anus, inserted opposite of the first branched dorsal-fin ray. Anus located 1.53-2.17 times eye diameter in front of anal-fin origin. Anal-fin rays iii, 5½. Caudal-fin rays 9+8, distal margin of caudal fin emarginated with upper and lower lobes almost equal in length. Moderate axillary pelvic lobe with free tip.
Body scaleless, or sparse scales scattered along lateral line after posterior end of anal-fiin base, embedded beneath skin. Lateral line completed with 85-89 pores. Supraorbital pores 7, postorbital pores 3, sub-and preorbital pores 12, preoperculo-mandibular pores 10, supratemporal pores 3. Vertebrae (three specimens), 4+37-39; four hypural elements with h-1 & h-2 fused, epurale present, last four neural spines (ns-1 to ns-4) and last three haemal spines (hs-1 to hs-3) on the caudal vertebrae are significantly enlarged. U-shaped stomach; intestine almost straight, with small bend next to stomach posterior. Longest recorded length is 83.1 mm SL, 95.7 mm total length (KIZ 2009000125, holotype).  Coloration of preserved specimens. Body light brown with vertical brown bars in formalin-fixed specimens. Bars on predorsal body usually blurred and indistinct, or countable and separated by extraordinary narrow interspaces just in KIZ 2009000122. Bars and interspaces getting wider towards caudal-fin base and approximately equal width on posterior body. Usually, bars regularly shaped and jointed on dorsal midline from opposite sides, or two bars met on upper body and last bar diffused or formed by two combined bars (KIZ 2009000122). Dark black bar on caudal-fin base, reaching dorsal and ventral extremities. All fin rays pale brown and covered by melanophores, series of stripes halfway up each dorsal-fin ray. Color in alcohol-fixed specimens is paler than those in formalin-fixed specimens.
Sexual dimorphism. No sexual dimorphism was observed. Etymology. Robusta is a Latin word meaning 'strong', in reference to the stout body and caudal peduncle. The Chinese common name is suggested as 粗壮荷马条鳅.

Discussion
Homatula wenshanensis was questioned as member of the genus Homatula by Nguyen et al. (2021), because of its indistinct adipose crests along the dorsal and ventral midlines of caudal peduncle, a forked caudal fin and 4+47-48 vertebrae that are not shared by species of Homatula. The results of our skeleton scan showed that H. wenshanensis has the typical generic character of Homatula -crests on caudal peduncle supported by rudimentary procurrent rays (Fig. 4C) -and our COI-based phylogeny showed that H. wenshanensis formed an independent lineage sister to H. disparizona. Therefore, H. wenshanensis is confirmed as a species of the genus Homatula.
Homatula is previously believed to be restricted to China. Recently, H. dotui, a cave-dwelling species, was reported from central Vietnam (Nguyen et al. 2021). Homatula dotui is between Schistura and Homatula as an independent lineage in the phylogenetic tree built by the cytb gene (Nguyen et al. 2021), which indicates that this cavefish species could belong to an undescribed genus. As stated by Nguyen et al. (2021: 8), a further study should be addressed to confirm the placement of H. dotui.
Three species of Homatula have been previously reported from the Nanpanjiang River: H. oligolepis and H. longidorsalis are distributed in the upper Nanpanjiang River; H. nanpanjiangensis is distributed in the middle Nanpanjiang River. They possess an elongate body of medium to large size, scaleless (H. oligolepis and H. nanpanjiangensis) or at least scaleless on the predorsal body (H. longidorsalis), 9 ½ branched dorsal-fin rays (H. oligolepis and H. longidorsalis) or 8 ½ (a few 9 ½ in H. nanpanjiangensis), regular vertical bars on each side of body, and bars in front of dorsal-fin base conspicuously thinner than those behind (H. longidorsalis and H. nanpanjiangensis) or vermiform markings on body and dorsal head (H. oligolepis). Here, H. robusta sp. nov. is reported from the middle Nanpanjiang River with a stout body. For better identification, a key to species distributed in the Nanpanjiang River is provided.
Key to species of Homatula in the Nanpanjiang River