Re-description of the loach species Leptobotia citrauratea (Teleostei, Botiidae), with the description of L. brachycephala from southern Zhejiang Province, China

Abstract Leptobotia citrauratea (Nichols, 1925), a loach species, originally described from Dongting Lake, was recently rehabilitated, based on the examination of the holotype and non-topotypical specimens. Several field surveys conducted from 2016 to 2019 in Zhejiang, Jiangxi and Hunan Provinces, P.R. China, yielded many specimens of Leptobotia which were initially identified as L. citrauratea. Molecular and morphological analyses of these specimens demonstrated that two distinct species are involved. One was identified as L. citrauratea, represented by specimens from both the Poyang and Dongting Lake (type locality) systems in Jiangxi and Hunan Provinces, and the other species is described as L. brachycephala, represented by specimens from the Ou-Jiang and Qu-Jiang, two coastal rivers of Zhejiang Province, China. Leptobotia brachycephala resembles L. citrauratea and L. micra in having a row of orange dots or an orange stripe along the dorsal mid-line of the body, extending from the nape to the caudal-fin base – a unique character in Leptobotia. Leptobotia brachycephala differs from L. citrauratea and L. micra Bohlen & Šlechtová, 2017, in caudal-fin shape and pelvic-fin insertion and proportional measurements including caudal-fin length, head length, predorsal length and anal-fin length. Its species status was further corroborated by position in a molecular phylogenetic analysis, based on the mitochondrial cyt b gene and its minimum uncorrected p-distance (2.9%) from congeneric species.


Introduction
The loach genus Leptobotia was erected by Bleeker (1870) with the simultaneouslydescribed Leptobotia elongata (Bleeker, 1870) as type species by monotypy. The genus is distinguished from other genera of the family Botiidae by the presence of a simple suborbital spine beneath the eye (Tang et al. 2008). Sixteen species are currently included in Leptobotia (Kottelat 2012;Šlechtová 2016, 2017). The majority of these species are known from southern China, mainly in the Yangtze River (= Chang-Jiang) and Pearl River (= Zhu-Jiang) Basins and coastal rivers of southern Zhejiang Province. Two species -L. flavolineata Wang, 1981 and L. orientalis Xu, Fang & Wang, 1981 -occur in northern China (Tang et al. 2008;Kottelat 2012;Bohlen and Šlechtová 2017). Nichols et al. (1925) described Leptobotia citrauratea from the Dongting Lake system in Hunan Province, China. Chen (1980) considered specimens of L. citrauratea to be juveniles of L. elongata. The synonymy of L. citrauratea with L. elongata was generally accepted by subsequent researchers (Kottelat 2004(Kottelat , 2012. However, Nalbant (2002) regarded L. citrauratea to be a valid species. Bohlen and Šlechtová (2017), based on examination of the holotype and non-topotypical specimens from the Poyang Lake system in Jiangxi Province, southern China, recognised L. citrauratea as a valid species. A row of orange dots or an orange stripe along the dorsal mid-line distinguishes it from L. elongata.
Several field surveys conducted by us from 2016 to 2019 in Zhejiang, Jiangxi and Hunan Provinces, yielded many specimens of Leptobotia with a row of orange dots or an orange stripe along the dorsal mid-line and orange or yellowish-brown lateral portion, by which they were initially identified as L. citrauratea. These specimens were recovered in two distinct lineages in a phylogenetic analysis, based on the mitochondrial cytochrome b (cyt b) gene sequences. Morphological analysis also indicated that two distinct species are involved. One of them was identified as L. citrauratea, represented by specimens sampled from the Poyang and Dongting Lake systems. The other species is an undescribed species represented by specimens from the Ou-Jiang and Qu-Jiang in Zhejiang Province. The present study aims to provide a re-description of L. citrauratea, based on fresh topotypical specimens and the formal description of the undescribed species.

Materials and methods
Specimens were either initially fixed in 10% formalin and then transferred to 70% ethanol for morphological examination or preserved in 95% ethanol for DNA extraction. Seventy-three specimens from the three species (L. citrauratea, L. elongata and L. brachycephala) were used for morphometric analysis. Voucher specimens are kept in the ichthyological collection of the Museum of Aquatic Organisms at the Institute of Hydrobiology (IHB), Chinese Academy of Sciences, Wuhan City, Hubei Province, China.
Twenty-five measurements (Tables 1, 2) were taken from 22 specimens of Leptobotia elongata collected from the upper Chang-Jiang Basin in Sichuan Province; 29 specimens of L. citrauratea from the Gan-Jiang (an effluent of Poyang Lake) and Dongting Lake; and 22 specimens of L. brachycephala from the Ou-Jiang and Qu-Jiang. Measurements were taken point to point with digital calipers directly linked to a datarecording computer and data recorded to the nearest 0.1 mm. All measurements and counts were made on the left side of each specimen, following the methods of Kottelat (2001) and Xin et al. (2009). The lateral head length and measurements of other parts of the body were given as percentages of the standard length (SL) and measurements of parts of the head were expressed as proportions of the lateral head length (HL). Morphometric variation was analysed with Principal Component Analysis (PCA) in Past v.1.89 (Hammer et al. 2009). The PCA was made with log-transformed measurement data to a tenth of a millimetre in a covariance matrix and without rotation.
Genomic DNA was extracted from fin clips stored in ethanol using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing) with the recommended protocol. The cyt b gene was amplified by primers L14724 (GACTTGAAAAACCACCGTTG) and H15915 (CTCCGATCTCCGGATTACAAGAC) adopted from Xiao et al. (2001), with 1 μl of each primer, 1 μl template DNA, 12.5 μl Master mix Taq (Beijing TsingKe Biotech Co. Ltd.) and 9.5 μl double distilled water (dd H 2 O) for a total reaction volume of 25 μl. The thermocycling conditions were as follows: initial denaturation for 4 min at 94 °C, denaturation for 50 s at 94 °C, annealing for 50 s at 55 °C and extension for 1 min at 72 °C. After 34-35 cycles, the final extension was done at 72 °C for 10 min and the PCR product was preserved at 4 °C. Sequencing was carried out by the Tianyihuiyuan Biotechnology Company.
A total of 98 cyt b sequences were generated from 12 species of Leptobotia. These sequences were used for phylogenetic analysis together with five sequences from two congeneric species (L. posterodorsalis Lan & Chen, 1992 and L. elongata) and two sequences serving as outgroup (Parabotia fasciata Dabry de Thiersant, 1872 andP. lijiangensis Chen, 1980) downloaded from GenBank (Table 3).
The sequences were aligned utilising MAFFT version 7 (Katoh and Standley 2013) and ends trimmed, for a total alignment length of 1060 bp. The genetic distance, based on the uncorrected p-distance model (Kumar et al. 2016), was calculated with MEGA 7.0. DNASP v.5 was utilised to filter the haplotype (Librado and Rozas 2009).
PhyloSuite (Zhang et al. 2020) was used for phylogenetic analyses. The selection of the best-fit model of nucleotide evolution based on Akaike's Information Criterion was performed in ModelFinder (Kalyaanamoorthy et al. 2017). MrBayes 3.2.6 (Ronquist et al. 2012) was utilised for Bayesian analysis with the selected model: GTR+I+G+F, applying the optimal nucleotide evolution model and the MCMC method with four chains (three hot chains and one cold chain) running simultaneously for 6,000,000 generations to calculate posterior probability. Trees were sampled for every 1000 cycles. The initial 25% of sampled data were discarded as burn-in. Sufficient mixing of the chains was regarded to be reached when the average standard deviation of split frequencies was below 0.01. Nichols, 1925 (Fig. 1a-c)  Fin rays flexible. Dorsal fin with 4 unbranched and 8 branched rays; distal margin slightly concave; origin slightly anterior to or superior to pelvic-fin insertion and closer to caudal-fin base than to snout tip. Pectoral fin with 1 unbranched and 10-11  branched rays, tip of depressed fin extending about midway between pectoral-fin and pelvic-fin insertion. Pelvic fin with 1 unbranched and 7 branched rays, reaching about half of distance between pelvic-fin insertion and anal-fin origin and just reaching anus. Anus closer to anal-fin insertion than pelvic-fin insertion. Anal fin with 3 unbranched and 5 branched rays, tip of depressed fin not extending to caudal-fin base; distal margin slightly concave. Caudal fin strongly forked, median fin rays 1.7-2.3 times as long as lobes; upper and lower lobes broadly pointed and almost equal in length and shape. Colouration. In freshly-collected specimens, ground colour of head and body yellowish-brown or orange; lateral head and flank faintly peppered with dark grey flecks. Dorsal side of head and body dark with some rounded light orange spots usually fused to form an orange stripe extending along mid-line of dorsum from nape to caudal-fin base. Anterior to orange spots or light stripe, an orangish stripe present between eye and nape. Faint dark grey stripe extending from snout tip to anterior margin of eye. Grey bar, similar in width to eye diameter, present on caudal-fin base. In some specimens, caudal fin hyaline, in others with dark grey stripes. Single row of faint dark grey stripes present in dorsal fin.

Leptobotia citrauratea
In specimens preserved in formalin, ground colour slightly faded, not presenting vivid yellowish-brown or orange, but becoming whitish-grey and peppered with dark flecks. Dorsum and head darkened. Orange spots along mid-line of dorsum white. Dorsal, pectoral, pelvic and anal fins greyish-yellow at base with white distal margins. Caudal fin dusky.
Description. Morphometric data given in Tables 1, 2. See Fig. 4 for lateral and dorsal view of body. Body slender, strongly compressed laterally, with greatest depth at dorsal-fin origin. Dorsal profile of head rising progressively from tip of snout to nape, from there to caudal-fin base nearly straight. Ventral profile of head slightly concave; ventral profile of body almost straight or slightly concave. Lateral line nearly complete, extending along mid-lateral body to terminate in median caudal-fin rays. Cheek and trunk covered with some minute scales.
Head short, compressed laterally, longer than maximum body depth. Snout slightly obtuse in lateral view, slightly shorter than postorbital head. Eye small, dorsolateral, in upper half of head; diameter less than interorbital width. Mouth inferior, with opening laterally extended to vertical through anterior margin of nostril. Button-like structures in gular region absent; no median incisions in lower lip. Two rostral barbels at tip of snout. Maxillary barbel in corner of mouth, not reaching to level of anterior margin of eye. Simple suborbital spine ventral to anterior margin of eye, not or just reaching posterior margin of eye.
Fin rays flexible. Dorsal fin with 4 unbranched and 8 branched rays; distal margin slightly concave; origin slightly posterior to pelvic-fin insertion and closer to caudal-fin base than to tip of snout. Pectoral fin with 1 unbranched and 10-11 branched rays, not extending to midway from pectoral-fin to pelvic-fin insertion. Pelvic fin with 1 unbranched and 7 branched rays, not extending to halfway to anal-fin origin or not reaching anus; vent closer to anal-fin origin than to pelvic-fin insertion. Anal fin with 3 unbranched and 5 branched rays, not reaching caudal-fin base; distal margin slightly concave; origin closer to pelvic-fin insertion than to caudal-fin base. Caudal fin emarginate or shallowly forked, length of median fin rays 1.3-1.5 times in length of upper lobe; caudal-fin lobes rounded; upper and lower ones almost equal in length and shape.
Colouration. In freshly-caught specimens, ground colour of head and body brownish-yellow; darker in upper half of head, but lighter in lower half of head and ventral side of body. A continuous or discontinuous orange stripe along mid-line of dorsum from nape to caudal-fin base, becoming more conspicuous towards caudal-fin base. Anterior to orange stripe, a short orange stripe present between eye and anterior margin of nape. A dark grey stripe on basal portion of dorsal fin and one stripe on dorsal fin. A dark grey band at caudal-fin base. Some irregular black stripes on caudal fin with hyaline distal edge. Distinct stripes absent from other fins. Specimens stored in formalin with ground colour of head and body pale brown. Discontinuous or continuous white line along dorsal mid-line of body also faded.
Geographical distribution and habitat. Leptobotia brachycephala is known only from the Ou-Jiang and Qu-Jiang, two coastal rivers of southern Zhejiang Province, China (Fig. 3). Type specimens were caught in fast-flowing clear water with mixed substrate including pebbles, gravels and boulders (Fig. 5) Explanation of name. The specific epithet is a Latin version of the Greek words βραχύς (short) and κεφαλά (head), with reference to the short head; to be treated as a noun in apposition.
Genetic comparisons. A total of 50 unique haplotypes were detected amongst the 103 cyt b sequences of species of Leptobotia (Table 3). The fragment contained 784 conserved sites, 276 variable sites, 233 parsimony informative sites and 43 singleton sites. The average frequency of four nucleotides of L. citrauratea was A = 27.8%, T = 27.8%, C = 30.3% and G = 14.1%. The intraspecific genetic distance, calculated for sampled species of Leptobotia with more than one haplotype, varied from 0.1% to 0.8%. Leptobotia citrauratea is separated from other congeneric species by high genetic divergences of 2.9% to 10.5%; its intraspecific genetic distance was 0.4%. The genetic  Comparative morphometrics. In the Principal Component Analysis of specimens of L. citrauratea from Dongting Lake and the Gan-Jiang and L. brachycephala from the Ou-Jiang and Qu-Jiang, the first three components explained 91.60% of the total variance, of which 64.58%, 19.61% and 7.41% were explained, respectively by PC 1, PC 2 and PC 3 (Table 5). In the scatterplot of PC 2 and PC 3 loadings (Fig. 7), specimens of L. citrauratea and L. brachycephala constituted two distinct clusters separated on the Table 4. Genetic distances of the cyt b gene computed by MEGA 7 amongst 13 analysed species of Leptobotia. PC 2 axis. Six characters with main loading on this axis were caudal-peduncle length, anal-fin length, dorsal-fin length, upper caudal-lobe length, pectoral-fin length and vent to anal-fin distance. Except for the last character, all of them exhibited differences in the morphometric comparisons. Table 2 and Fig. 2 show the main morphological characters.  Nichols et al. (1925) described the colour of L. citrauratea as "purplish brown; yellowish below". This colouration is shared with freshly-caught specimens of this species from the Dongting Lake (type locality) and Poyang Lake systems (Fig. 1). Our examination of these topotypical specimens and a photograph of the holotype (AMNH 8402) (Fig. 1c) confirmed Bohlen and Šlechtová's (2016) observation that L. citrauratea has some round orange dots or an orange stripe along the dorsal mid-line of the body. There were no significant differences in morphometric measurements and meristic counts detected between specimens from the Dongting and Poyang Lake systems (Table 1). Chen (1980) synonymised L. citrauratea with L. elongata, a species found in the mid-upper Chang-Jiang Basin, but without examination of their type specimens or even reference to topotypical specimens. This classification had been widely accepted by subsequent researchers until 2002 when Nalbant recognised L. citrauratea as valid. Morphological data, provided in this study, indicated that there were distinct variations between these two species. Topotypical specimens examined of L. citrauratea possessed a small-sized body of up to 70.0 mm SL, while the body size of L. elongata attained a length of 97.8 to 272.0 mm SL for available specimens caught from the upper Chang-Jiang Basin. The specimen of the species, caught by Fang (1936), reached up to 500 mm in total length. Leptobotia citrauratea has a series of small orange spots or an orange stripe along the dorsal mid-line of the body from the nape to the caudal-fin base, a yellowish-brown or orange ground colour of head and body and no black band crossing the dorsum (Fig. 1). This is contrast to L. elongata which, in light of its original account and our observation on specimens collected from the upper Chang-Jiang Basin, has a body colouration of many wide brown and transverse bands (Fig. 8a, b). Additionally, L. citrauratea differs from L. elongata in having larger eyes (diameter 9.2-12.1% HL vs. 4.2-8.4% HL; see Table 1) and pelvic fin not or just reaching (vs. exceeding) the anus. Our molecular analysis also showed that L. citrauratea had a 7.1% interspecific genetic distance with L. elongata (Table 4) and that these two species constituted two independent lineages distantly related in the phylogenetic tree, based on the cyt b gene (Fig.  6). It is thus concluded here that L. citrauratea is a species distinct from L. elongata and confined only to the mid-lower Chang-Jiang Basin (the Dongting and Poyang Lake Basins); L. elongata is actually an endemic species of the upper Chang-Jiang Basin.  Nichols et al. (1925) recognised three species of Botia from the Dongting Lake system: one previously-described species as Botia rubrilabris Dabry de Thiersant, 1872 and two new species, B. purpurea and B. citrauratea. These three species were later referred to Leptobotia where B. purpurea were synonymised with L. taeniops (Sauvage, 1878) (Chen 1980;Kottelat 2004Kottelat , 2012. The latest report on the distribution of L. rubirilabris (Dabry de Thiersant, 1872) in this Lake was Anonymous (1980) who caught a single specimen of 80.0 mm SL. This specimen, in light of their description, has a buttonlike fleshy protrusion in the gular area, a character diagnostic for L. rubirilabris within this genus (Chen 1980); thus, it is conspecific with this species. Recent field surveys, conducted by us from 2014 to 2018 in Dongting Lake, yielded no specimens of this species. Likely, it was extirpated in this Basin. Leptobotia citrauratea mainly differs from L. rubrilabris, caught from the upper Chang-Jiang Basin (its type locality), in having a shorter (vs. longer) suborbital spine just reaching (vs. far beyond) the posterior margin of the eye and no button-like fleshy protrusion in the gular area (vs. present) (Fig. 9).

Discussion
The specific status of L. brachycephala was confirmed by its morphological and genetic distinction with closely-related congeneric species (Tables 2-4). The PCA results showed that specimens of this species from southern Zhejiang Province formed a cluster, distinct from the one formed by specimens of L. citrauratea from the Dongting and Poyang Lake systems (Fig. 7). Although L. brachycephala was robustly supported by 100% pp to be sister to L. citrauratea, their interspecific genetic distance was 2.9%.
The seventeen species currently included in Leptobotia can be subdivided into six groups, based on their body colourations. The first one is only composed of one species L. taeniops that has a unique body colouration of some irregular purplish-brown stripes in the shape of a worm on the flank, hence resulting in a marbled or vermiculated pattern (Fig. 8c). The second group, represented by L. punctata Li, Li & Chen, 2008, has a lot of irregularly-organised white spots on the flank, giving a reticulated pattern. The third group, including L. pellegrini Fang, 1936, L. elongata, L. hengyangensis Huang & Zhang, 1986, L. tchangi Fang, 1936 and L. rubrilabris, is characterised by having a body colouration of some broad brown-black blotches on the body or saddles on the dorsum. The fourth group is formed by the following four species: L. posterodorsalis, L. bellacauda Bohlen & Šlechtová, 2016, L. tientainensis (Wu, 1930) and L. microphthalma Fu & Ye, 1983, all of them having a plain brown body and no other colour formation. The fifth group has some rounded light orange spots extending along the mid-line of the dorsum from nape to caudal-fin base and three species are included in this group: L. micra, L. citrauratea and L. brachycephala. The last group has narrow bands on the body or mid-line of the back and three species can be referred to this group: L. guilinensis Chen, 1980, L. orientalis and L. flavolineata.