﻿Oreonectesdamingshanensis (Cypriniformes, Nemacheilidae), a new species of stream fish from Guangxi, Southwest China

﻿Abstract In this work, a new species of the genus Oreonectes is described, named Oreonectesdamingshanensis Yu, Luo, Lan, Xiao & Zhou, sp. nov., collected from the Damingshan Mountains of the Guangxi Zhuang Autonomous Region, China. Phylogenetic trees constructed based on the mitochondrial Cyt b showed that the new species represents an independent evolutionary lineage, with uncorrected genetic distances (p-distance) from congeners ranging from 6.1% to 8.9%. Morphologically, the new species can be distinguished from five other species of the genus by a combination of characters. The discovery of this new species raises the number of known species of Oreonectes from five to six. Our study suggests that O.platycephalus may be a complex containing multiple species and that previously recorded areas need to be further delimited and reevaluated.


Introduction
A globally important biodiversity hotspot, the karst region of southwest China is characterized by extremely high species diversity and endemism (Wang et al. 2018).Rivers and mountains as geographic isolation are potential environmental drivers of species formation and diversification (Antonelli et al. 2018), which is also reflected in freshwater fishes from the karsts of southwest China (Wen et al. 2022).Thus, the unique geomorphological and hydrological conditions of karst may have led to the isolation of different geographical populations of widely distributed species, resulting in speciation (Jiang et al. 2022;Li et al. 2022).The large number of new fish species found in the karst region of southwest China over the past decade (Lan et al. 2013;Zhang et al. 2019) also suggests that the species diversity of its freshwater fishes may have been underestimated.Therefore, researchers are required to carry out detailed field surveys for taxonomic studies to assess the potential biodiversity of the area.
Between 2020 and 2023, we collected 30 specimens during field surveys of open streams in Nanning City, Guangxi Zhuang Autonomous Region, China (Fig. 1).Through examination of these specimens and molecular sequencing, we observed significant differences in both morphology and mitochondrial cytochrome b gene (Cyt b) in these specimens when compared with their congeners.These differences led us to identify this as a new species of the genus Oreonectes, which we named Oreonectes damingshanensis sp.nov.

DNA extraction, PCR, and sequencing
Genomic DNA was extracted from muscle tissue using a DNA extraction kit from Tiangen Biotech (Beijing) Co. Ltd.Two muscle samples of the new species were sequenced for the mitochondrial cytochrome b gene (Cyt b).The forward and reverse primers used for Cyt b were F14724 (5'-GACTTGAAAAAC-CACCGTTG-3') and R15915 (5'-CTCCGATCTCCGGATTACAAGAC-3'), respectively, following Xiao et al. (2001).PCR amplifications were performed in a 25 μl reaction volume with the following cycling conditions: an initial denaturing step at 95 °C for five min, 36 cycles of denaturing at 95 °C for 40 s, annealing at 45 °C for 40 s and extending at 72 °C for 1 min, and a final extension at 72 °C for 10 min.The purified products were sequenced with both forward and reverse primers using a BigDye Terminator Cycle Sequencing Kit according to the manufacturer's instructions.The products were sequenced on an ABI Prism 3730 automated DNA sequencer by Chengdu TSING KE Biological Technology Co. Ltd. (Chengdu, China).All sequences have been deposited in GenBank (Table 2).

Phylogenetic analyses
A total of 41 Cyt b sequences were used for phylogenetic analysis.In addition to the four new sequences, the remaining 37 sequences were downloaded from GenBank and included five already recognized genera (Table 2) and two outgroup species from the mitogenome provided by Luo et al. (2023).
Mitochondrial Cytb sequences were aligned in MEGA v7.0 (Kumar et al. 2016) by the MUSCLE (Edgar 2004) algorithm with default parameters.Phylogenetic trees were constructed using both maximum likelihood (ML) and Bayesian inference (BI) methods.The ML tree was conducted in IQ-TREE v2.0.4 (Nguyen et al. 2015) with 2000 ultrafast bootstrap (UFB) replicates (Hoang et al. 2018) and was run until a correlation coefficient of at least 0.99 was reached.The BI phylogeny was constructed in MrBayes v3.2.1 (Ronquist et al. 2012).Two independent runs were conducted in the BI analysis, each of which was performed for 2 × 10 7 generations and sampled every 1000 generations.The first 25% of the samples was discarded as a burn-in, resulting in a potential scale reduction factor of < 0.01.For BI and ML analyses, the best-fit model was obtained based on the Bayesian information criterion computed with PartitionFinder v2.1.1 (Lanfear et al. 2017).In this analysis, the first, second, and third codons of the Cyt b gene were defined.The results of the model selection suggested that the first, second, and third codons of the best-fit model for the Cyt b gene were K80+I+G, HKY+I+G, and TRN+I+G, respectively.Nodes in the trees were considered well supported when Bayesian posterior probabilities (BPP) were ≥ 0.95 and the ML ultrafast bootstrap value (UBP) was ≥ 9 5%.Uncorrected p-distances (1000 replicates) based on the Cyt b gene were calculated using MEGA 7.0 (Kumar et al. 2016).

Morphological comparisons
Morphometric data were collected from 53 well-preserved specimens of the genus Oreonectes (Appendix 3: Table A1).A total of 33 measurements were recorded to the nearest 0.1 mm with digital calipers following the protocol of Tang et al. (2012).All measurements were taken on the left side of the fish specimens.
Comparative data for the five species of the genus Oreonectes were obtained from the literature and specimen examination (Table 4).Specimens of four species from the type locality were examined, including O. guananensis, O. luochengensis, O. platycephalus, and O. polystigmus (see Appendix 1).Considering the morphological similarity of the new species to O. platycephalus and O. polystigmus, the measurements were also included in the statistical analysis.Principal component analyses (PCAs) of size-corrected measurements and simple bivariate scatterplots were used to explore and characterize the

X-ray scanning and three-dimensional image reconstructions
In order to obtain information on the skeletons of the new species, X-ray scanning was conducted via nano-computerized tomography.Specimens were scanned using a GE v|tome|x m dual tube 300/180 kv system at the Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Chinese Academy of Sciences.Each specimen was scanned with an energy beam of 80 kV and a flux of 80 μA using a 360° rotation, and the data were then reconstructed into a 4096 × 4096 matrix of 1536 slices.The final CT reconstructed skull images were exported with a minimum resolution of 6.099 μm.The skull images were exported from the virtual 3D model and reconstructed by Volume Graphics Studio 3.0.

Phylogenetic analyses and genetic divergence
BI and ML analyses were performed to construct phylogenetic trees with consistent topologies based on mitochondrial Cyt b sequences with a length of 1140 base pairs (Fig. 2).These phylogenetic trees showed a topology similar to that of Luo et al. (2023), but with lower node support between major clades (Fig. 2).In addition, several new clades were identified, and Yunnani- Distance between anterior and posterior nostrils 0.6-1.5 0.9 ± 0.2   2.
lus was divided into two distant clades named Yunnanilus (I) and Yunnanilus (II).K. anophthalmus, K. parva, and K. acridorsalis were clustered together to form a separate clade.In fact, all five remaining species of the genus Oreonectes, clustered together to form a sister clade of the genus Lefua.
Within the genus Oreonectes, the four specimens collected from Mashan and Shanglin counties in Nanning City, Guangxi, China, formed a distinct and highly supported clade with O. platycephalus and O. guilinensis (0.99 in BI and 85% in ML) (Fig. 2).Within the genus Oreonectes, the genetic distances between the new species Oreonectes damingshanensis sp.nov.and the remaining five species range from 6.1% (for O. polystignus) to 8.9% (for O. guananensis).This level of divergence was similar to those between pairs of other recognized species.For example, the Cyt b p-distance was 4.9% between O. luochengensis and O. guananensis (Table 3).

Morphological analyses
Mann-Whitney U tests showed that the Oreonectes damingshanensis sp.nov.differed from O. luochengensis, O. polystignus, O. guananensis, and O. platy-cephalus in several morphological characters (Table 4).These significant differences were mainly observed in the head, fins, and tail regions.The differences were more pronounced in comparisons of the new species with O. platycephalus, with 84.8% of the morphological characters being significantly different (p = 0.00−0.046)(Table 4).Based on PCA of the morphological data, two principal component factors with eigenvalues greater than one were extracted.These accounted for 84.09% of the total variation (Appendix 4: Table A2).The first principal component (PC1) accounted for 77.92% of the variation and was positively correlated with all variables (eigenvalue = 28.91),thus reflecting the morphological differences between Oreonectes damingshanensis sp.nov.and similar species.This axis corresponded to body length, head, fins, nostrils, and barbel length.Thus, based on the statistical analysis of the measurements and the PCA and CDA results, 30 specimens from Oreonectes damingshanensis sp.nov.were clearly distinguished via morphological characters from the four similar species O. luochengensis, O. polystignus, O. guananensis, and O. platycephalus.The second principal component (PC2) accounted for 6.17% of the variation and was influenced by the length of the distance between posterior nostrils, length of the upper jaw, mouth width, and eye width (eigenvalue = 0.04) (Appendix 4: Table A2).The two-dimensional plots of PC1 and PC2 clearly separated Oreonectes damingshanensis sp.nov.from O. polystignus and O. platycephalus (Fig. 3A).CDA correctly classified 100% of the individuals in the initial grouping case for the three sample groups (N = 40).Canonical axes (CAN) 1-2 explained 75.8% and 24.2% of the total variation (Fig. 3B; Appendix 4: Table A2).
Diagnosis.Oreonectes damingshanensis sp.nov. is assigned to the genus Oreonectes based on molecular phylogenetic analyses and the following characteristics, which are diagnostic for this genus: (1) anterior and posterior nos- Description.Body elongated and cylindrical, with the insignificant depth decreasing from dorsal-fin origin to caudal-fin base.Head short, length 13.3 mm, slightly depressed and flattened, width greater than depth (head width/head depth = 1.3).Snout round, oblique and flat, length 38.0-45.3% of head length (HL).Mouth inferior, curved, upper and lower lips smooth, lower lip with a V-shaped median notch.Three pairs of barbels are long: inrostral barbel length 25.9-38.3% of HL, extending backward and not reaching the anterior margin of eye; outrostral barbel length 42.4-62.4% of HL, extending backward beyond the posterior margin of the eye.Maxillary barbel length 36.0-56.6% of HL, tip of maxillary barbel not reaching to posterior margin of gill cover.Anterior and posterior nostrils narrowly separated, length 33.5-84.2% of eye diameter.Anterior nostril tube short, with an elongated short barbel-like tip.Eyes present, normal, diameter of 6.5-17.1% HL.Gill opening small; gill rakers not developed, nine inner gill rakers on the first gill arch (n =1) (Fig. 4D).Dorsal-fin rays iii-7, pectoral-fin rays i-9, pelvic-fin rays i-7, anal-fin rays iii-5, 14 branched caudal fin rays.Dorsal fin short, length 15.8-22.6% of SL, distally margin round, origin posterior to pelvic-fin insertion, situated slightly posterior to two-thirds the distance between snout tip and caudal-fin base.Pectoral fin short, length 15.2-19.5% of SL, tip of pectoral fin extending backward to ~56.7% of the distance between the origin of pectoral and pre-pelvic fins.Pelvic fin length 13.1-17.2% of SL, tips of pelvic fin not reaching anus, distance between tips of pelvic fin and anus 2.0 times the eye diameter.Anal fin long, length 15.3-179.7% of SL, tips of anal fin not reaching caudal-fin base, distance between tips of anal fin and anus 0.64 times the eye diameter.Caudal fin rounded, caudal peduncle length 9.3 mm, without adipose crests along either dorsal or ventral sides.Vertebrae 38 comprising of 4 Weberian + 22 abdominal + 11 caudal + 1 compound centrum (Fig. 5).
Except for the head, body completely covered by scales.Lateral line incomplete, exceeding tip of pectoral fin but not reaching base of caudal fin, with 14 or 15 pores, last lateral line pore reaching above the tip of pectoral fin.Cephalic lateral-line system, with eight supraorbital, 5 + 8 infraorbital canal pores, three supratemporal canal pores, with eight preoperculo-mandibular canal pores.Two chambers of air-bladder, posterior chamber developed, filling the body cavity and connected with anterior chamber by a long, slender tube.Lateral wall of the bony capsule of the swim bladder is membranous and closed posteriorly (Fig. 5).
Coloration.In life, body pale yellow-brown overall, dark brown between anterior margin of eyes to outrostral barbel, brown lateral stripe on flank of the body, irregular black spots present on dorsal and lateral surfaces and caudal fin, black at base of caudal fin, pale brown on each fin (Fig. 6).In 10% formalin, the body color was dark brown (Fig. 4).
Comparisons.Comparative data of Oreonectes damingshanensis sp.nov.with the five known species within the genus Oreonectes are given in Table 1.(vs 11), seven branched pelvic-fin rays (vs 8), anal-fin rays (iii, 5 vs ii, 6 or 7), nine inner gill rakers on the first gill arch (vs 14 or 15), and maxillary barbel extending backward, not reaching to the posterior margin of the gill cover (vs reaching to the posterior margin of the eye).
Distribution, habitat, and populations.Based on current field surveys, the new species Oreonectes damingshanensis sp.nov.has only been found in streams in the Damingshan Mountains of Mashan and Shanglin counties in the Guangxi Zhuang Autonomous Region of China belonging to the Hongshuihe River basin of the Pearl River system.The new species is easy to collect in the streams of the Damingshan Mountains area where it has a large population.2).This may be related to the number and length of molecular markers and the number of species.In the phylogenetic tree reconstructed by Luo et al. (2023) based on the mitochondrial genome and seven nuclear genes, O. platycephalus (No. 27, i.e., Oreonectes damingshanensis sp.nov.) from the Damingshan Mountains was considered to be a sister species of O. polystignus, which was strongly supported by the BI tree but not fully resolved in the ML tree.This may be related to the absence of O. guilinensis.Therefore, the inclusion of a sufficient number of sampled species and molecular markers in future studies may help to resolve interspecific phylogenetic relationships within the genus Oreonectes.

Key to species of the gneus
The widely distributed species O. platycephalus Günther, 1868 may be a complex containing multiple cryptic species.O. platycephalus was originally described from specimens collected from small streams near the top of Hong Kong Mountains, the type locality (Günther 1868), and it has been extensively recorded from various tributary areas of the Pearl River Basin (the Luofu Mountains, the Baiyun Mountains and eastern Guangdong, and Jinxiu, Rong'an, and Zhaoping counties in Guangxi), including areas in northern Vietnam (Fig. 1) (Zhu 1989;Kottelat 2001;Zhang et al. 2016).The wide geographical distribution, morphological similarity, and classification based on morphological characteristics of species from the same basin may lead to different geographical populations of the species being identified as a single species, resulting in an underestimation of species diversity.The combination of mitochondrial differences and morphological characteristics of O. guilinensis supports this hypothesis (Huang et al. 2020).Furthermore, the small-bodied, widely distributed species of Oreonectes may rapidly form new species due to the geographical isolation of water systems, as shown in Fig. 1 and the phylogenetic tree (Fig. 2).Therefore, we recommend that a comprehensive and systematic survey (especially concerning the grey shaded area in Fig. 1) of O. platycephalus be carried out to assess its species diversity.
In the newly described species Oreonectes damingshanensis sp.nov.and the other five species, O. guananensis and O. luochengensis are the most northerly distributed and have adjacent distributions and phylogenies, followed by O. polystigmus and finally O. guilinensis and O. platycephalus.By combining the positions of these species in the phylogenetic tree, we propose the hypotheses of "neighboring and closely related" and "northern Guangxi origin with southward dispersal", i.e., within the genus Oreonectes, the shorter the distance between species, the more closely related they are in the phylogenetic tree, and the earlier the species formed at the northern tip of their distribution.These "neighboring and closely related", "microlocalism", and "north to south" patterns of geographic distribution and phy-

Figure 1 .
Figure 1.Sampling collection localities and distribution of Oreonectes damingshanensis sp.nov.and five species of the genus Oreonectes in southern China.For details of ID numbers, please see Table 2.The green rectangular box without an ID is the type locality of O. platycephalus.The base maps are from the Standard Map Service website (http://bzdt.ch.mnr.gov.cn/index.html).

Figure 2 .
Figure 2. Phylogenetic tree based on mitochondrial Cyt b (1140 bp).In this phylogenetic tree, Bayesian posterior probabilities (BPP) from BI analysis/ultrafast bootstrap supports (UFB) from ML analysis are listed beside nodes.The scale bar represents 0.08 nucleotide substitutions per site.The numbers at the tip of branches correspond to the ID numbers listed in Table2.

Figure 3 .
Figure 3. Plots from the principal component analysis, and canonical discriminant analysis scores of Oreonectes damingshanensis sp.nov., O. polystignus, and O. platycephalus based on morphological characters.

Figure 4 .
Figure 4. Morphological characteristics of the holotype GZNU20230216001 of Oreonectes damingshanensis sp.nov. in preservative (10% formalin) A lateral view B dorsal view C ventral view D gill raker E enlarged anterior and posterior nostrils.

Figure 5 .
Figure 5.The three-dimensional reconstructed model of the skeleton of Oreonectes damingshanensis sp.nov.(paratype GZNU20230216021, standard length 57.0 mm) A dorsal view B ventral view, and C latera view.Caudal fin slightly broken.

Table 1 .
Species list of the genus Oreonectes and comparisons of diagnostic characters of the new species with congeners.Grey shading indicates a clear difference in a character compared to that of Oreonectes damingshanensis sp.nov.

Table 2 .
Localities, voucher information, and GenBank numbers for all samples used.

Table 3 .
Uncorrected p-distances (%) between five species of the genus Oreonectes based on the mitochondrial Cyt b gene.
(Parsons and Jones 2000;Polaszek et al. 2010)sidered statistically significant at a p-value < 0.05.PCAs of morphological data were performed after logarithmic transformation and under conditions of no rotation.In addition, as reported by other researchers(Parsons and Jones 2000;Polaszek et al. 2010), canonical discriminant analysis (CDA, George and Paul 2010) was used to classify individuals into different groups, where a priori membership was determined based on specimens belonging to different species.All pre-processing of morphological data was performed in Microsoft Excel (Microsoft Corporation 2016).

Table A1 .
ZooKeys 1180: 81-104 (2023), DOI: 10.3897/zookeys.1180.104645Jing Yu et al.: Oreonectes damingshanensis, new species logeny suggest that Oreonectes is a good candidate for speciation and biogeographic studies in the Pearl River Basin.To test this hypothesis, future studies could focus on population genetics and biogeographic study of the O. platycephalus complex.Measurements of the five specimens of Oreonectes damingshanensis sp.nov.and other species.All units in mm.*designates the holotype.

Table A2 .
Results and percentage of variance explained by principal component and discriminant function analyses.