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 soft-tissue 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) and Zhang et al. (2019), respectively. Specimens examined are deposited in the Museum of Aquatic Organisms at the Institute of Hydrobiology (IHB), Chinese Academy of Sciences, Wuhan City, Hubei Province, China (Suppl. material 1). The suffixes -Jiang, -Shui and -He mean river or stream in Mandarin Chinese.

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 (Zhang et al. 2020). 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.

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.

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). From the tree topology, samples of this new species constituted a strongly-supported (100% posterior probabilities and 100% bootstrap values) independent lineage that was distantly related to the lineage formed by topotypical samples of V. stenosoma from the Yong-Jiang, but nested with the lineage constituted by the sample (SCAU0926783), under the name of V. stenosoma, from Qu-Jiang into a clade (hereafter called V. aff. stenosoma) sister to the clade made of by samples from the following five species: V. maculata, V. intermedia, V. stenosoma, V. hainanensis and V. polylepis.

Figure 5.  

Phylogenetic tree reconstructed using Bayesian method (BI) and Maximum Likelihood (ML), based on cyt b gene. Values at the nodes correspond to the support values for BI/ML methods.

Vanmanenia marmorata had the minimum genetic distance of 11.3% with V. stenosomaand the maximum one of 15.4% with V. gymnetrus (Table 4). The mean genetic distance of the new species with all sampled Chinese congeneric species was 13.0%, far greater than the minimum ones (2.6%), here detected between V. pingchowensis and V. homalocephala. The new species had a significant sequence divergence from V. maculata and V. pingchowensis (12.2–15.2%). The intraspecific genetic divergence of V. marmorata was 0.2%.

The new species can be assigned to the group characterised by having three triangular-shaped 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 lateral-line scales (78−90 vs. 95−100). Vanmanenia gymnetrus was transferred to the fourth group, defined by Yi (2014) as having multiple barbel-like forms derived from the apical parts of the rostral lobules, as well as secondary rostral barbels. This group is the same as ‘the barbelled rostral fold group’ defined by Li et al. (2019) as having three rostral lobes modified into papillae and secondary barbels. V. gymnetrus is further distinct from V. marmorata in having more lateral-line scales (92−104 vs. 78−90), a slender (vs. stout) caudal peduncle (depth 0.63−0.67 vs. 0.72−1.01% of length) and a narrower (vs. wider) interorbital space (width 35.7−37.0 vs. 37.3−41.0% of HL).

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%).