Vanmaneniamarmorata, a new species of loach (Teleostei: Gastromyzontidae) from the middle Chang-Jiang Basin in Guizhou Province, south China

Abstract Background The gastromyzontid genus Vanmanenia was established by Hora in 1932, based on the type species Vanmaneniastenosoma. The genus is a loach group adapted to running waters of streams from southern China, northern Vietnam and Laos. Currently, 19 valid species of the genus have been recognised. The northernmost distribution of the genus is the Yangtze River (= Chang-Jiang in Chinese) Basin and five species (V.maculata, V.intermedia, V.stenosoma, V.pseudostriata and V.gymnetrus) have been reported from the Basin. New information Vanmaneniamarmorata, a new hillstream species of loach, is here described from the middle Chang-Jiang Basin in Guizhou Province, south China. It is distinguished from its congeners by having a combination of the following characters: three triangular-shaped rostral lobules; postdorsal saddles wider than interspaces; a more backwards-placed anus (the vent to anal distance 30.5–36.9% of the pelvic to anal distance); a larger gill opening with its upper extremity reaching the level of the middle of the orbit; anal-fin base length 5.6–6.4% of SL; caudal-peduncle length 11.6–12.9% of SL; prepelvic length 51.1–53.4% of SL. Its validity is also affirmed by its distinct cyt b gene sequence divergence with all sampled congeners and its monophyly recovered in a cyt b gene-based phylogenetic analysis.


Introduction
The gastromyzontid genus Vanmanenia Hora, 1932, a group of loaches, occurs widely in southern China, northern Vietnam and Laos (Kottelat 2012). It has specialised rostral barbels and lips and was placed in the Crossostomini-group, a special branch in the evolution of the Gastromyzontidae (Chen 1980, Tang and. The genus is distinguished by having a trilobated rostral fold and two pairs of rostral barbels; lower lip with four large papillae and postlabial groove short and restricted only to mouth corner; gill opening extending ventral surface of head . Currently, a total of 22 species have been identified in this genus, including three Vietnamese species whose taxonomic status is urgently needed to be confirmed: V. nahangensis Nguyen 2005, V. multiloba (Mai 1978 and V. ventrosquamata (Mai 1978) (Fricke et al. 2021). Three valid species (V. crassicauda Kottelat 2000, V. caobangensis Nguyen 2005 andV. orcicampus Kottelat 2017) are found only in the Red River Basin in Vietnam and the Mekong River Basin in Laos (Li et al. 2019, Deng and. Vanmanenia has 16 representatives in south China where they are now known from the Yuan-Jiang (=Red River), Lixian-Jiang, Lancang-Jiang, Chang-Jiang, Zhu-Jiang, Han-Jiang, Jiulong-Jiang, Min-Jiang, Ou-Jiang, Ling-Jiang, Qiantang-Jiang and Changhua-Jiang and Wanquan-He of Hainan Island (Tang and , Li et al. 2019, Deng and Zhang 2020. Amongst them, there are 13 endemic Chinese species (Li et al. 2019, Deng and Zhang 2020, Fricke et al. 2021. The northernmost distribution of Vanmanenia is the Yangtze River (= Chang-Jiang in Chinese) Basin in which five species have been presently identified: V. maculata, V. gymnetrus, V. intermedia, V. stenosoma and V. pseudostriata (Yi et al. 2014, Li et al. 2019, Zhang et al. 2019, Deng and Zhang 2020, Wang and Zhang 2021. All specimens of Vanmanenia from this Basin had been reported as V. pingchowensis (Ding 1994, Zhu 1995 until 2014 when Yi et al. (2014) described V. maculata from the Qing-Jiang and Li-Shui (in the middle Chang-Jiang Basin). Later, Zhang et al. (2019) named a new species V. pseudostriata, based on specimens previously misidentified by Zhou et al. (2010) as V. pingchowensis from the Zhangjiu-He, a stream tributary to the main stem of the Jinsha-Jiang (in the upper reaches of the Chang-Jiang Basin) in Luquan County, Yunnan Province. Recently, Deng and Zhang (2020) revalidated V. intermedia from the synonym of V. pingchowensis, concluding that the former is present in the Qingshui-Jiang of the upper Yuan-Jiang of the middle Chang-Jiang Basin and the Hongshui-He of the middle Zhu-Jiang Basin. Wang and Zhang (2021) also documented V. stenosoma and V. gymnetrus from the Gan-Jiang, an effluent of Lake Poyang (in the lower Chang-Jiang Basin).
A sixth species of Vanmanenia from the middle Chang-Jiang Basin in Guizhou Province, south China is the one described in the present study. Field surveys of fishes conducted during June 2017 into the Yuan-Jiang, an effluent of Lake Dongting, yielded many specimens referrable to Vanmanenia. In comparing these specimens, they turned out to be two distinct species. The one is represented by V. intermedia and the other does not conform to any currently-identified congeneric Chinese species, therefore representing an unnamed species. The specific status of this unnamed species, here described as V. marmorata, is further affirmed by molecular phylogenetic analysis.

Materials and methods
All measurements were taken point-to-point with a dial caliper and recorded to the nearest 0.1 mm. Morphometric data (see Table 1) and meristic counts were made on the left side of individuals when possible, following Yi et al. (2014). Prepectoral, prepelvic, predorsal and pre-anal lengths were measured from the tip of snout to the pectoral-, pelvic-, dorsal-and anal-fin origins, respectively. Caudal-peduncle length was taken from anal-fin insertion to the last vertebra. Morphometric measurements were expressed as percentages of standard length (SL) or of head length (HL). The numbers of fin rays were counted under a stereoscopic microscope. The terms utilised in this study for description of mouth part softtissue structures and their definitions also follow Yi et al. (2014). Data used here for V. gymnetrus, V. intermedia, V. maculata andV. pseudostriata come from Chen (1980), Deng and Zhang (2020), Yi et al. (2014)  The genomic DNA was extracted from the right-side pelvic-fin tip and mitochondrial cytochrome b (cyt b) gene was selected for amplification and sequencing. The primers and the PCR reaction followed Xiao et al. (2001) and Deng and Zhang (2020), respectively.
Amplified products were subsequently purified and utilised for sequencing by a commercial sequencing company. The obtained sequences were spliced using Seqman from DNASTAR's Lasergene (Burland 2000) and then checked by utilising BLAST analysis in GenBank database. After confirming, the targeted sequences were submitted to GenBank database.
We sequenced cyt b gene of V. stenosoma, V. homalocephala, V. hainanensis, V. caldwelli and V. marmorata and retrieved the cyt b gene sequences for other species of Vanmanenia from GenBank (Table 2). Pseudogastromyzon tungpeiensis and Erromyzon kalotaenia were selected as outgroups. All sequences were aligned by MAFFT 7.0 (Katoh and Standley 2013) in PhyloSuite ). The genetic distances (p-distance with 1,000 bootstrap) of the two sequences between taxa were calculated by utilising MEGA 7.0 (Kumar et al. 2016). The best substitution model (GTR+F+I+G4) was selected in ModelFinder (Kalyaanamoorthy et al. 2017) by Akaike's Information Criterion (AIC). The phylogenetic trees were inferred using Bayesian Inference (BI) and Maximum Likelihood (ML) approaches. Bayesian analyses was conducted using MrBayes (Ronquist et al. 2012). Four simultaneous Monte Carlo Markov chains run for 1 million generations, with sampling one tree per 100 replicates for each run and the first quarter of the trees were discarded as burn-in; the remaining trees from two independent runs were used to construct a consensus tree. The ML analyses were conducted using IQ-TREE (Nguyen et al. 2015) with a total of 10,000 bootstrap replicates performed.

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 shown in Table 2.

Description
Morphometric measurements for specimens examined are given in Table 1. See Fig. 1 a-c for lateral, dorsal and ventral view of body.
Body elongate and slightly compressed, with greatest depth at dorsal-fin origin and least depth at caudal-fin base. Dorsal profile of head rising abruptly before, then increasing evenly towards dorsal-fin origin, from there, to caudal-fin base decreasing gradually. Lower surface of head straight and oblique; ventral profile of body from pectoral-fin insertion to anal-fin origin slightly concave, oblique along anal-fin base and straight in caudal peduncle. Head moderately depressed, slightly longer than wide and wider than high, with slightly broad and convex interorbital space. Snout slightly rounded in dorsal view. Eyes small, dorsolateral in posterior half of head. Anterior and posterior nostrils separated and short flap on anterior ones. Mouth small, inferior and arched. Rostral fold divided into three triangular-shaped lobules, median one slightly wider than two lateral ones; tips of these lobules in barbel-like form, but not modified into secondary rostral barbels (Fig. 2). One pair of maxillary barbels at corners of mouth; two pairs of rostral barbels in deep groove present between rostral fold and upper lip. Upper lip pendulous and connected with lower lip around corners of mouth by papillated flap; lower lip with four large papillae, two median ones more anteriorly placed and usually larger than the two lateral ones; postlabial groove short and restricted only to mouth corner. Upper and lower jaws bearing thick, with flexible horny sheaths on flanks. Gill opening reaching ventral surface of head, with its upper extremity reaching the level of the middle of the orbit. Body scaled, but absent on head; scales minute, lateral-line complete with 78-90 perforated scales. Anus placed significantly nearer anal-fin origin than pelvic-fin origin.
Dorsal fin with 3 unbranched and 7-8 branched rays, nearly as long as head; dorsal-fin origin almost located halfway between caudal-fin base and snout tip; distal margin slightly concave. Pectoral fin with 1 unbranched and 13-14 branched rays, longer than head; inserted slightly behind lower extremity of gill-opening; tip of adpressed rays not reaching pelvic-fin insertion; distal margin convex. Pelvic fin with 1 unbranched and 8 branched rays, inserted slightly closer to caudal-fin base than to snout tip or midway between pectoral-fin insertion and anal-fin origin or slightly moved backwards; tip of adpressed rays surpassing anus, but far from anal-fin origin; distal margin pointed; axillary lobe present at pelvic-fin base. Anal fin with 2 unbranched and 5 branched rays, last one split to base; origin closer to caudal-fin base than to pelvic-fin insertion; distal margin convex. Anus positioned closer to anal-fin origin than to posterior end of pelvicfin base. Anal fin extending beyond ventral origin of caudal-fin procurrent rays. Caudal fin slightly forked with lower lobe slightly longer than upper one.  Coloration: In alcohol-stored specimens, top of head with numerous, small, irregular, black bars and blotches; yellowish on ventral surface of head and abdomen. Body with 7-9 dorsal dark black saddles. Last predorsal, subdorsal and first postdorsal saddles wider than all others. Postdorsal saddles wider than interspaces. A number of irregular black blotches on flank above lateral line, nearly connected with dorsal saddles. These black blotches extended downwards to ventral surface of body. Blotch through pelvicfin insertion extended downwards to base of axillary lobe at pelvic-fin base. A black spot on caudal-fin base, darker than markings on flank. Dorsal fin with 3 black bands across rays. Anal fin with 1 black band. Pectoral fin with 3 irregular faint black bands across rays. Pelvic fin with up to 3 black bands. Caudal fin with 4 black bands.

Diagnosis
Vanmanenia marmorata resembles the four species ( V. caldwelli, V. maculata, V. intermedia and V. stenosoma) in having three triangular-shaped rostral lobules whose apical portions are in the barbel-like form, but not modified into secondary rostral barbels. It is distinct from these species in having postdorsal dark black saddles wider (vs. narrower) than their interspaces, further from V. caldwelli in having no longitudinal black stripe extending from the snout tip to the caudal-fin base along the lateral line on flank (vs. present) and a more backwards-placed anus [the vent to anal distance 30.  Table 3); and from V. intermedia in having a larger gill opening with its upper extremity reaching the level of the middle of the orbit (vs. smaller, closer to the level of the lower margin of the orbit; see Deng and Zhang 2020 : Page 117: fig. 2), a shorter (vs. longer) anal-fin base [length 5.6-6.4 (mean 6.0) vs. 7.5-9.5 (mean 8.3) % of SL) and a longer (vs. shorter) caudal peduncle [length 11.6-12.9 (average 12.0) vs. 8.4-11.1 (average 9.9) % of SL]; and from V. stenosoma in having a longer (vs. shorter) caudal peduncle [length 11.6-12.9 (mean 12.0) vs. 9.0-11.1 (mean 10.0) % of SL] and a more forwards-positioned pelvic fin [prepelvic length 51.  Table 3.

Etymology
The specific epithet is from the Latin word marmor referring to the unique body colouration of irregular marbled markings.

Distribution
This new species is presently known from the upper reaches of the Chen-Shui, a stream tributary to the Yuan-Jiang of the Dongting Lake system in the middle Chang-Jiang Basin, at Jiangkou County, Guizhou Province, south China (Fig. 3). It inhabits fast-flowing waters with a gravelly and pebbly substrate (Fig. 4). Co-existing species are Discogobio yunnanensis (Regan, 1907), Onychostoma barbatum (Lin, 1931) and Rhinogobius cliffordpopei (Nichols, 1925).

Sequence variation and molecular phylogeny
Thirty-one cyt b gene sequences of Vanmanenia were used for phylogenetic analysis. After alignment and trimming, 1095 bp (base pairs) of the cyt b gene were obtained. There were 735 conserved sites, 360 variable sites, 268 parsimony informative sites and 92 singleton sites. The mean frequency of four nucleotides was A=25.9%, T=29.3%, C= 29.6% and G= 15.2%; the base composition was A-T rich (55.2%).
The two analysis methods (BI and ML) showed an identical topology (Fig. 5)

Discussion
The new species can be assigned to the group characterised by having three triangularshaped rostral lobules whose apical parts are in the barbel-like form and connected with the rostral lobule, but not modified into secondary rostral barbels, based on the grouping of Chinese species of Vanmanenia by Yi (2014). Four species are, so far, identified in this group, namely V. intermedia, V. maculata, V. stenosoma and V. caldwelli (see the diagnosis for detailed differences of this new species with these four species).
Vanmanenia marmorata is presently known only from the Yuan-Jiang of the middle Chang-Jiang Basin. In addition to V. intermedia, V. maculata and V. stenosoma, there are two other congeneric species V. pseudostriata and V. gymnetrus also found in the upper and lower Chang-Jiang Basin, respectively. Within Vanmanenia, V. pseudostriata was assigned to either the barred group defined by Li et al. (2019) as having vertical black bars on flank and spotless paired fins or the first group by Yi (2014) as having three rounded rostral lobules. These three characters can separate the new species from V. pseudostriata with vermiculated marks on the flank, up to three bands across the rays of paired fins and three triangular-shaped rostral lobules. The new species is further distinct from V. pseudostriata in possessing a more backwards-set anus (closer to the anal-fin origin than to the pelvic-fin insertion vs. midway between the pelvic-fin insertion and anal-fin origin) and less lateralline scales (78−90 vs. 95−100). Vanmanenia gymnetrus was transferred to the fourth group, defined by Yi (2014)  The validity of V. marmorata is corroborated by its remarkable genetic divergence from sampled congeners (Table 4) and its monophyly recovered in the phylogenetic analysis, based on the cyt b gene (Fig. 5). The new species had a 0.2% intraspecific genetic divergence and a significant genetic divergence with sampled congeneric Chinese species (11.3-15.4%), far greater than the minimum calculated here between V. pingchowensis and V. homalocephala (2.6%). The topology of the phylogenetic tree also showed that the new species was sister to V. aff. stenosoma, represented by the sample (SCAU0926783) previously misidentified as V. stenosoma from the Qu-Jiang. The type locality of V. stenosoma is Ningbo, southern Zhejiang Province, in the Yong-Jiang, a coastal river flowing into the South China Sea. Two topological samples of V. stenosoma were highly supported to unite with samples of the paired species (V. maculata and V. intermedia) into an lineage, and distantly related to the sample (SCAU0926783), under the name of V. aff. stenosoma, from the Qu-Jiang. This sample (SCAU0926783) might represent an undescribed species distinct from this new species, given a clear barcode gap between the paired species (11.9%).