Homatula guanheensis sp. nov. (Teleostei: Nemacheilidae), a new species of loach from Henan Province, China

Abstract Background The genus Homatula belongs to the order Cypriniformes and family Nemacheilidae. Nichols (1925) set up the genus as a subgenus of Barbatula by the type species of Nemacheilus potanini. Currently, it is recognised as a valid genus. Nineteen valid species have been already reported in the drainage of the Yellow, Yangtze, Pearl, Lancang, Red and Nujiang Rivers. H. variegata, H. longidorsalis, H. berezowskii and H. potanini are distributed in the Yangtze River drainage in China. H. laxiclathra is mainly distributed in the Weihe River, a tributary of the Yellow River. The remaining species are mainly distributed in the rivers of Yunnan Province. New information Homatula guanheensis sp. nov., a new species, is described from the Guanhe River of the HanJiang River drainage (a tributary of the Yangtze River), Xixia County, Henan Province, China. It can be distinguished from its congeners by a combination of the following characters: the vertical brown bars on the body are wider than their interspaces, numbering 19–22; predorsal body partially scaled; the lateral line complete; adipose crest on caudal peduncle not reaching forward; the position of the anal-fin origin and the intestinal form. The new species displays distinct molecular divergence in the Cytochrome oxidase I (COI) and Cytochrome b (Cyt b) genes.


Introduction
The Nemacheilid genus Homatula was established by Nichols in 1925 as a subgenus of Barbatula, based on the type species Nemacheilus potanini. Species from Homatula are small-size benthic fishes that are distributed in the drainage of the Yellow, Yangtze, Pearl, Lancang, Nujiang and Red Rivers. Some researchers have treated Homatula as a synonym of Paracobitis according to its adipose-crest (Chu and Chen 1990, Ding and Deng 1990, Min et al. 2010, Zhou and He 1993, Zhu 1989, Zhu and Cao 1988, Zhu and Wang 1985. However, the type species of Paracobitis was described from western Asia and these had a large geographical gap with the Chinese species (Bǎnǎrescu andNalbant 1995, Kottelat 1990). Therefore, we accept the opinion of species in western Asia as Paracobitis and all species in China as Homatula (Kottelat 1990, Bǎnǎrescu andNalbant 1995). Nine species of Homatula have been reported from China before 2010. Homatula erhaiersis (Zhu and Cao, 1988) was treated as a synonym of H. anguillioides (Zhu and Wang, 1985) in recent studies by the comparison of phylogenetic analysis and morphological characteristics , Endruweit et al. 2018). In addition, an increasing number of new species of Homatula have been reported in recent years: H. disparizona (Min et al. 2013) was the first recorded species of Homatula from the Red River; H. wenshanensis (Yang et al. 2017) and H. coccinocola (Endruweit et al. 2018) were also found in the Red River; H. change (Endruweit 2015) was found in the upper Black River Basin; H. wuliangensis ) and H. pycnolepis (Hu and Zhang 2010) were collected from the Lancang River drainage; H. nanpanjiangensis (Min et al. 2010) was reported from the Pearl River; H. laxiclathra (Gu and Zhang 2011) was found in the Wei-He River of the Yellow River drainage in Shaanxi Province and three new species of Homatula were identified in the upper Salween River (Li et al. 2019).
The morphological variation of Homatula in the upper Yellow River drainage, the uppermiddle reaches of Yangtze River drainage and the upper Pearl River drainage was investigated by Zeng et al. in 2012. However, the taxonomic status of the specimens from the Hanjiang River of the middle Yangtze River drainage were undetermined (Zeng et al. 2012). The specimens of Homatula from the Guanhe River (a tributary of the Han River) were collected during the investigation of fishery resources in Xixia County, Henan Province. Morphological data and DNA taxonomy (COI and Cyt b) were used to identify the samples and the analysis found differences with the species that had been previously discovered. An unnamed species, different from congeners, is described in this paper.

Materials and methods
All specimens were examined and stored in the collection of the College of Fisheries, Henan Normal University, Xinxiang, Henan Province, China. For morphological analysis, 10 specimens were fixed in 10% formalin. All measurements and counts were made following Kottelat (1990). Measurements were made point to point with digital calipers to the nearest 0.1 mm. X-ray films were used to count simple fin rays. Comparative morphometry was examined using PCA in IBM SPSS Statistics 22.0. The PCA analysis was processed using log-transformed morphometric data of percent of Standard Length (SL) in the variance-covariance matrix. To compare molecular characters, eighteen specimens were fixed in 95% ethanol and total genomic DNA was extracted from muscle tissue using standard phenol-chloroform extraction protocols (Sambrook et al. 1989). Two pairs of primers were used to amplify segments of COI and Cyt b by the polymerase chain reaction. The COI primers were designed with reference to other Osteichthyes COI gene sequences: Fish-CO I-F (5'-TCT CAA CCA ACC ATA AAG ACA TTGG-3'); Fish-CO I-R (5'-TAT ACT TCT GGG TGC CCA AAG AAT CA-3'). The Cyt b gene primers were designed as GluF (5'-AAC CAC CGT TGT ATT CAA CTA CAA-3'); ThrR (5'-ACC TCC GAT CTT CGG ATT ACA AGA CCG-3') (Machordom and Doadrio 2001). PCR reactions were carried out in a 30 μl volume and amplifications proceeded for 5 min at 94°C followed by 34 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 60 s and final extension at 72°C for 10 min. The PCR products were sent to a commercial corporation for sequencing. The nucleotide sequences were assembled from independent sequence passes using the SeqMan (Swindell and Plasterer 1997) module of the DNAStar software package. Additionally, sequences for other species of Homatula were obtained from GenBank (Endruweit et al. 2018, Xiong et al. 2017, Yue et al. 2013. Thirteen species of Homatula were studied, from which 680-bp COI sequences of 50 samples and 1,120-bp Cyt b sequences of 42 samples were used to identify the new species. A list of taxa with corresponding GenBank accession numbers is presented in Table 1. Phylogenetic analysis was performed using PhyloSuite (Zhang et al. 2020). Multiple alignments were performed with MAFFT version 7 with the default parameters (Katoh and Standley 2013). Then, ModelFinder (Kalyaanamoorthy et al. 2017) was used to ascertain the best-fit model of nucleotide substitution for the sequences using the AIC. Tree analyses were performed under Bayesian Inference (BI). All Bayesian phylogenetic analyses were conducted with MrBayes 3.2 (Ronquist et al. 2012), based on the GTR+F+G4 model estimated by ModelFinder. Four Markov chains were run for 1,000,000 generations to estimate the posterior probability distribution, sampling every 1000 generations. After discarding the first 1000 trees as a burn-in with non-stationary log likelihood values, 50% majority-rule consensus trees were estimated for the remaining trees. Finally, all sequences were grouped according to the results of the phylogenetic analysis. The genetic distances between groups were calculated using MEGA7.0 (Kumar et al. 2016), based on the K2P model.

Data resources
All the sequences in this study were retrieved from GenBank and the accession numbers of the newly determined sequences in this study are MT771689-MT771705 (COI) and MT771706-MT1722 (Cyt b).  (Table 2). Mouth inferior, lips thick and furrowed, jaws covered by lips, upper jaw with developed processus dentiformis corresponding with marked median notch on lower jaw. Three pairs of barbels: two rostral barbels, inner pair not reaching mouth corner and outer pair reaching vertical line of anterior nostril; one maxillary barbel extending to the middle and posterior margin of eye (Fig. 1). Dorsal fin ⅲ, 7-8 rays, origin nearer to snout tip than to caudal-fin base. Pectoral fin ⅰ, 9-10 rays, not extending beyond halfway from its origin to the pelvic-fin origin. Pelvic fin ⅰ, 6-7 rays, its origins closer to vertical line of first branched rays of dorsal fin; tip of the pelvic fin not extending beyond half the distance from its origin to anal fin origin. Anal fin ⅲ, 5 rays; origin of anal fin closer to pelvic fin origin than to caudal fin base, its tip not reaching half distance from anal-fin origin to caudal-fin base. Posterior margin of caudal fin micro-rounded; adipose crests along its dorsal and ventral mid-lines without extending through the origin of anal fin. Intestine formed as a bend, not reaching posterior surface of the U-shaped stomach. Gas bladder osseous, anterior chamber invisible, fully enclosed in a capsule; posterior chamber degenerated.    Color in preserved specimens (fixed in 10% formalin): Head and body brown; a series of 19-22 body bars, each bar at least twice as wide as the interspace. Abdomen yellowish. Dorsal fin with two dark brown marks, one at the base, the other at postmedian of the fin; posterior border of dorsal fin white. Pectoral fins with dark brown spots. Pelvic and anal fins white, dark at the base. Adipose keels white with dark brown spots. Caudal fin dark grey; brown vertical bars on caudal fin base (Fig. 1).

Diagnosis
Homatula guanheensis is different from its congeners in the following characters: partly scaled (  Scatter plots of the first and second principal components from morphometric data of H. guanheensis (n = 10, 76.9 -109.26 mm SL) and H. laxiclathra (n = 23, 67.6-136.7 mm SL).

Etymology
The specific epithet Guanheensis is derived from Guanhe River (鹳河 in Chinese, type locality) with the Latin suffix "-ensis".

Distribution
Homatula guanheensis sp. nov. is known from the Guanhe River of the Hanjiang River drainage (a tributary of the Yangtze River) in Henan Province, Central China (Fig. 3).  nov.

Genetic distance and phylogentic trees
Based on the COI gene, sequences of 11 studied species were used to construct a BI tree. The new species belonged to a different clade with strong high PP values (100%, Fig. 4), the genetic distance being analysed by the Kimura 2-parameter model. The distance matrix and number of sequences, determined for each species, are shown in Table 4 and Table 5. Genetic distance between H. guanheensis and other given species displayed 4.7%-10.6% variation in the COI base pairs (Table 4). The smallest distance, 2.9% (5 vs. 9) was detected between H. pycnolepis and H. anguillioides. Based on the Cyt b gene, the phylogenetic tree analysis showed that H. guanheensis is a monophyletic group with a high posterior probability value (100%; Fig. 5). Through the results of the genetic distance analysis, we found that H. guanheensis differed from the acquired congeners by 4.2%-10.3%. The genetic distance between H. guanheensis and similar species exceeded 2% variation, while the intraspecific variation of H. guanheensis was 0% ( H. coccinocola (2) 5 0.0 8.0 7.1 7.0 9.7 6.5 8.9 6.9 4.9 11.9   The phylogenetic tree of Homatula based on the Cyt b gene; Schistura latifasciata was used as the outgroup.  (Zhu 1989, Yang et al. 1994, Hu and Zhang 2010 Fig. 5), but the new species and H. longidorsalis + H. variegata form a sister group (supported by the COI gene, Fig. 4). The cause might vary amongst datasets and, thus, this discordance might be influenced by stochastic errors associated with a different number of species and datasets (Naylor and Brown 1998). The results of Cyt b data and the COI data show that the new species has a far genetic distance from H. berezowskii, H. variegata and H. longidorsalis.