Sinobdellalongitubulus, a new species of spiny eel (Pisces, Mastacembelidae) from the Zhu-Jiang Basin, with a note on the type locality of S.sinensis (Bleeker, 1870)

Abstract Background The spiny eel genus Sinobdella belongs to the family Mastacembelidae of the order Synbranchiformes. Kottelat and Lim (1994) utilised Rhynchobdellasinensis as the type species to propose the genus. Currently, it contains a single species widespread in eastern and southern China and northern Vietnam. New information Sinobdellalongitubulus, a new species of spiny eel, is here described from the Xi-Jiang of the Zhu-Jiang Basin in Guangxi Zhuang Autonomous Region, southern China. It differs from the single congeneric species S.sinensis in having a more or less white-brown reticulated pattern on the flank, two tubular anterior nostrils longer than or equal to the rostral appendage, an anal fin heavily mottled with dark brown markings and white spots and bearing a narrow white distal margin; shorter pre-anal length; and fewer abdominal vertebrae. The validity of this new species is corroborated by its monophyly recovered in a COI gene-based phylogenetic analysis and its significant sequence divergence with S.sinensis. A note on the type locality of S.sinensis is also given; its type specimen is possibly from mountain streams of Jiangxi Province, in the lower Chang-Jiang Basin.


Introduction
The family Mastacembelidae, widely known as spiny eels, consists of anguilliform percomorph fishes, characterised by continuous dorsal spines along the mid-line dorsal body.These species are extensively distributed in tropical Africa, the Middle East and South and Southeast Asia, where they inhabit diverse freshwater environments such as lakes, streams and rivers, feeding on zooplankton, aquatic insect larvae and small fish (Nelson et al. 2016).Amongst three genera to date identified in the family (Roberts 1986, Vreven andTeugels 2005), Sinobdella is the only monotypic genus spanning from the Liao-He, an independent river flowing into Bohai Sea, in Liaoning Province (Xie 2007), through eastern and southern China (including Taiwan Island) (Shao 2022), to the Red River in northern Vietnam (Kottelat 2001, Kottelat 2013).
The generic placement and specific status of the single species S. sinensis (Bleeker 1870a) have ever remained contentious.It was initially described in Rhynchobdella Bloch & Schneider and later referred to either Mastacembelus Scopoli (Wang 1984, Wang et al. 2001) or Pararhynchobdella Bleeker (Zhu 1995).This spiny eel belongs to its own genus (Kottelat and Lim 1994).In Chinese literature , this species had been synonymised with Mastacembelus aculeatus (Basilewsky, 1855) before it was viewed as valid (Zheng 1989, Liu 1991, Zhou 1997, Tang et al. 2001).
The taxonomy of S. sinensis is still poorly understood due to a lack of information on its precise type locality.The original description stated that this species was from China, but without specifying its accurate location (Bleeker 1870a).This poses a hindrance to in-depth taxonomic investigations on the species diversity of S. sinensis, so leading to a taxonomic inertia of this spiny eel since its original description.It has widely been regarded as a widespread species in China (Zhang et al. 2016).However, our ongoing taxonomic study of this species from China demonstrates significant morphological and genetic differentiations between the Pearl River (= Zhu-Jiang in mainland China) and Yangtze River (= Chang-Jiang) Basin populations.Our textual research revealed that its type locality is mountain streams of Jiangxi Province, in the lower Chang-Jiang Basin (see explanation in the Discussion section).Clearly, the true S. sinensis is represented by the Chang-Jiang Basin population.Many specimens, caught during 2021 to 2023 by us from the Meng-Jiang (in Pingnan and Mengshan Counties) and Hongshui-He (in Shanglin County) of the Zhu-Jiang Basin in China, belong to an unidentified recognised species.The objectives of the present study are to: (1) provide a detailed description of this unidentified species and (2) clarify the uncertainty concerning the type locality of S. sinensis.

Specimen sampling, preservation and curation
The specimens of Sinobdella used in this study were obtained via field sampling, in accordance with the Chinese Laboratory Animal Welfare and Ethics animal welfare laws (GB/T35892-2018).Some caught individuals after being anaesthetised were stored in 10% formalin for morphological observation and permanent curation, following the removal of a small flip of pectoral fin preserved in 95% ethanol.Other individuals were directly preserved in 95% ethanol or 10% formalin after being anaesthetised.Examined specimens are housed in the Museum of the Institute of Hydrobiology (IHB), Chinese Academy of Sciences, Wuhan City, Hubei Province, P. R. China, British Museum of Natural History (BMNH) and the Muséum national d'Histoire naturelle (MNHN).

Morphological analysis
Measurements were taken point to point with a digital caliper linked directly to a datarecording computer and data recorded to the nearest 0.1 mm.Measurements were made on the left side of specimens whenever possible.The counts of vertebra, dorsal spine and dorsal-and anal-fin rays were taken from X-ray photographs.Pectoral-and caudal-fin rays are difficult to count in a dissecting scope and, thus, not included here.All counts and measurements follow Vreven and Teugels (1996).If the number of fin rays and vertebrae could not be counted in a given X-ray photograph, the part of this photograph will be magnified until it can be clearly counted.Measurements of head were expressed as percentages of lateral head length (HL) and HL and measurements of other parts of body were given as proportions of standard length (SL).GraphPad Prism 9 (GraphPad Prism Inc.) was utilised for the basic statistical analysis of morphometric measurements.

Molecular analysis
Total genomic DNA was extracted from the pectoral fin stored in 95% ethanol using the TIANamp Genomic DNA Kit (Tiangen Biotech Co., Ltd, Beijing, China) following the manufacturer's recommendations.The mitochondrial cytochrome C oxidase subunit I (COI) gene was chosen for phylogenetic analysis.Amplification and sequencing of the target gene were achieved using the COI-F1 primer (5′GTGGCAATCACACGTTGAT′3) (Jiang 2018), along with a primer specifically designed for this study, COI-R1 (5′ATGGAGGTTCGATTCCTTC′3). The target gene was amplified by polymerase chain reaction (PCR) in a 25 μl mixture, consisting of 1 μl of each primer, 12.5 μl of Taq Master mix (Genesand Biotech Co. Ltd., Beijing, China), 1 μl of template DNA and 9.5 μl of double-distilled water (ddH O).The PCR was executed under the following thermocycling conditions: an initial denaturation at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 53°C for 40 seconds and extension at 72°C for 1 minute and 30 seconds, with final extension at 72°C for 8 minutes.The PCR products were subsequently stored at 4°C.Sequencing was performed by Aokedingsheng Biotechnology Company (Wuhan, China) and the sequences obtained in this study were submitted to GenBank.

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 Suppl.material 1.
Morphometric data for two species of Sinobdella from China.

Range
Second anal spine placed close to first spine, but distinctly distant from third spine, much longer than both.

Diagnosis
Sinobdella longitubulus is clearly distinguished from the single congeneric species (S. sinensis) by having a more or less white-brown reticulated pattern (vs.many dark brown vertical bars, with very narrow light yellow interspaces) on the flank (Fig. 2), two tubular anterior nostrils longer than or equal to (vs.shorter than) the rostral appendage (Fig. 3), an anal fin heavily mottled with dark brown markings and white spots and bearing a narrow white distal margin (vs.black with a relatively wide light white distal margin) (Fig. 2); shorter pre-anal length (53.3-56.2 vs. 56.3-60.6 % SL; see Fig. 4) and fewer abdominal vertebrae (32-33, mean = 32.9 vs. 34-36, mean = 35.1)(Table 1).

Etymology
The epithet name, used here as a noun, is derived from the Latin word longus (= long) and tubulus (= pipe), alluding to two longer tubes modified from anterior nostrils.The common Chinese name here suggested for this new species is "长管华刺鳅".

Colouration
In formalin-preserved specimens (Fig. 1), top of head whitish-brown and cheek and opercula brown, with a reticulated pattern of brown markings on ventral surface of head.A broad light yellowish mid-dorsal band extending from behind head to soft dorsal-fin origin and two narrow whitish stripes parallel to this band laterally, wider and  lightened anteriorly and prolonging backwards under soft dorsal-fin base.Lateral body brown numerous small white spots, more or less inconspicuous on upper twothirds of abdomen and caudal region and conspicuous and restricted only to lower onethird of abdomen to form a white-brown reticulated pattern, continued by a broad extension across belly.Pectoral fin pale, dorsal and caudal fins dark grey and anal fin dark grey with a narrow whitish distal margin and a dark black submarginal stripe.
In freshly-、collected specimens (Fig. 2a), dorsum of head and body yellowish-brown from snout tip to origin of soft dorsal fin, with two black longitudinal zigzag lines parallel to dorsal mid-line on each side, more or less connected to constitute a reticulated pattern of markings.Lateral body dark brown with numerous irregular white spots scattering densely over abdomen and sparsely on caudal region, more or less distinct and restricted to lower half of flank to form a reticulated pattern of markings.Continued dorsal and caudal fins light yellowish-brown with an indistinct reticulated pattern.Pectoral fins yellowish or orange and anal fin heavily mottled with dark brown markings and white spots, bearing a narrow light white distal margin and a very narrow submarginal black stripe.
Given that Bayesian Inference (BI) and Maximum Likelihood (ML) analyses produced overall identical topologies, only the BI tree with Bayesian posterior probabilities (PP) and bootstrap support (BS) value were provided in Fig. 7. From the tree topologies, samples of S. longitubulus were strongly supported (PP = 1.0/BS = 100) to cluster into a lineage further constituting a well-supported (PP = 1.0/BS = 100) clade together with S. sinensis.
The genetic distances (p-distances) within and between species were calculated.Intraspecific genetic distance for sampled species of S. sinensis and S. longitubulus were 0.5% and 0.1%, respectively.Interspecific genetic distance between both were 10.5%.

Discussion
Bleeker (1870a) described Rhynchobdella sinensis, based on a single 199 mm TL specimen caught in China.Despite no indication of the accurate type locality in the original description, Bleeker (1870b) noted that this species was one of 44 species then identified from Chinese specimens predominantly collected from the Yang-Tse-Kiang (or Yangtze River = Chang-Jiang in mainland China) and associated Lake Po Yang (= Lake Poyang) and Kan-Kiang River (presently Gan-Jiang, a stream of Lake Poyang) and the waters of Ning-Po (presently Ningbo).He further stated that these Chinese specimens, received from the administration of Jardin royal des plantes médicinales (the predecessor of the Muséum national d'Histoire naturelle, MNHN), constituted part of the collections by C.P. Dabry de Thiersant, G.E. Simon and A. David intended for the Museum in Paris (MNHN); the board of the Museum in Paris invited him to identify them.Apparently, the type specimen of S. sinensis ought to be housed at MNHN rather than at BMNH (British Museum of Natural History) and the collector of the type is likely one of the three persons mentioned.
There are seven records of Chinese spiny eels currently housed in MNHN (Table 2).Seven specimens [MNHN-IC-0000-5230 (two), 5573 (four) and 7490 (one)] of unknown source were collected from China by A. David in 1869 and 1870, respectively.Four specimens [MNHN-IC-0000-5021 and 5022 (two), from the Yangtze River (or today's Chang-Jiang); 7851 (one, from mountains of Jiangxi Province); and 7358 (one, from China, but without precise locality) were respectively collected by C. P. Dabry de Thiersant in 1863, 1868 and 1873.A comparison of X-ray photographs of two Chinese spiny eels reveals that Sinobdella sinensis has more vertebrae between the first and second anal spine pterygiophore than Mastacembelus armatus (four or five vs. one or two) (Fig. 8).In terms of our examination on the X-ray photographs available in MNHN, the first and second pterygiophore of anal spines are separated by one or two vertebrae in two specimens (MNHN-IC-0000-5021 and 5022) (Fig. 9b, c), so indicating their misidentification of M. armatus.The specimen (MNHN-IC-0000-7490) is not the type of S. sinensis as it was collected in 1870, the same year as the publication of its original account, but slightly after the collection time (1868) of the type specimen of this species (see explanation below).
The possibility is ruled out that the specimen (MNHN-IC-0000-7358) is the type of S. sinensis as it was caught in 1873, well after the publication of its original description.It is probable that the type of this species is amongst those caught by A.   from the Chang-Jiang Basin.The specimen (MNHN-IC-0000-7851) from the mountain streams of Jiangxi Province is most likely the type as it agrees with the original description in, amongst others, body size.
Chinese spiny eels under the name of S. sinensis in the BMNH collection are also amongst mastacemblids examined by both Travers (1984a), Travers (1984b) and Vreven (2005).Three specimens (BMNH 1888.3.23: 60-62) of 119-169 mm TL and one specimen (BMNH 1895.5.31: 13-14) of 202 mm TL, which were donated by F. W. Styan, according to information available in this Museum, are from Kiu-Kiang (= Jiujiang City), Jiangxi Province (in the lower Chang-Jiang Basin) and in Shanghai (in the estuary of this river), respectively.Although three specimens BMNH 1888.3.23:60-62 were listed as types (Travers 1984a) or syntypes (Vreven 2005), they are unlikely the types of S. sinensis as they disagree with its original description in body size, far less than 199 mm TL of the type, as well as the collection time as indicated by their catalogued number, well after the publication of its original description.The specimen BMNH 1895.5.31:13-14 was listed as a syntype with a question mark (Vreven 2005), possibly due to its similar body size, close to 199 mm TL given in the original description for the type.It is not the type of this species because of its later collection time, as indicated by its catalogued number.
The discovery of this new species from the middle Zhu-Jiang Basin in Guangxi Province necessitates a re-diagnosis of S. sinensis and a delineation of its distribution in China.
Characters typical for S. sinensis are given in the diagnosis.According to these characters, this species is extensively known from the mid-lower Chang-Jiang Basin.Populations of river basins north of this river, such as the Huai-He, Hai-He and Luan-He are conspecific with the species, as evidenced by molecular data (Fig. 7).This spiny eel is also found in the Li-Jiang and Luoqing-Jiang, two stream tributaries of the middle Xi-Jiang of the Zhu-Jiang Basin.The plausible explanation for this is that the spiny eel possibly dispersed into the two rivers from the Xiang-Jiang via the Ling Canal built in 214 BC.
Except for the count of vertebrae (Table 3), no distinct morphological and genetic variations are found between populations of the middle Zhu-Jiang and mid-lower Chang-Jiang Basin for S. sinensis.Sixteen specimens examined from Lingchuan County and Guilin City (in the Li-Jiang) and Yongfu County (in the Luoqing-Jiang) had 76-77 (mean 76.42) vertebrae, slightly fewer than 77-79 (mean 78) of four specimens examined from nearby Xing'an County (in the Xiang-Jiang, Guangxi Province; 76-78 (mean 77.6) of ten specimens from the Xiang-Jiang and Yuan-Jiang, two streams of Lake Dongting in Hengdong and Mayang Counties, Hunan Province; and 76-81 (mean 79.11) of 21 specimens collected from the Han-Jiang, the largest tributary of the Chang-Jiang Basin, in Xiangyang City and She-Shui in Dawu County, Fu-He in Sui County and Huan-He in Xiaochang County, three streams on the northern bank of the middle Chang-Jiang mainstream in Hubei Province.This variation can be plausibly explained by Jordan's rule that stipulates a geographical tendency for populations of the same fish species from higher latitudes to have an increased number of vertebral centra (Jordan 1891, McDowall 2007).
This phenomenon is also observed for S. sinensis in east China.For example, four specimens caught from the Qiantang-Jiang Basin in Changshan County, Zhejiang Although Kottelat (2013) ascribed it to Sinobdella, this generic assignment remains indeterminacy as this genus is currently unknown from the Mekong River Basin.Endruweit (2014) retained this spiny eel in Mastacembelus and confirmed that its four type specimens have been lost in RIA 1(Research Institute for Aquaculture No. 1, Bac Ninh, Hanoi).For this reason, its validity and generic placement remain yet to be resolved, only if topotypical specimens become available.For the time being, this species is excluded from Sinobdella.
Mastacembelus kobayashii was firstly described by Oshima (1926) from Taiwan Island.This spiny eel is only known by two type specimens in the original description.No additional specimens have been collected so far; its validity has remained yet to be resolved.Taiwanese spiny eels were previously identified as Macrognathus aculeatum (Bloch, 1786) (=Sinobdella sinensis) (Tzeng 1986).It has been considered as a subspecies of S. sinensis in the annotated checklist and type catalogue of fish genera and species described from Taiwan by Ho and Shao (2011).This taxonomic treatment is followed in this study.
region relatively short.Pre-anal length slightly greater than postanal length.Body covered with tiny scales without lateral line.Head short and pointed, with snout produced into fleshy and long rostral appendage projecting from upper jaw.Two tubular anterior nostrils located on underside of rostral appendage, longer than or equal to this appendage.Posterior nares oval, horizontal axis longest and situated in front of eyes.Eyes small, laterodorsally located at anterior half of head.Posteriorly growing single pre-orbital spine on right/left side of head, located under eyes and buried under skin, with ending point extending beyond anterior margin of eye, but not reaching middle of eye.Mouth inferior and horse-shoe-shaped.Lips thick and fleshy.Jaws with numerous small, pointed teeth; upper jaw longer than lower jaw.Angle of jaws between the posterior edge of the posterior external nare and the anterior edge of the eye.Preoperculum unarmed.Upper extremity of gill opening immediately above dorsal end of pectoral-fin base, slightly anterior to vertical through ventral end of pectoral-fin base.Dorsal fin long and divided into two parts: dorsal spines and dorsal-fin.Dorsal spines 32 to 33, increasing in size from first to second last spine; last spine smaller than second last spine, situated anterior to base of first soft dorsal-fin ray.Soft dorsal-fin rays 50-60.Anal spines 3 and soft anal-fin rays 50-60; anterior end of dorsal spines posterior to pectoral-fin insertion and anterior end of soft dorsal-fin rays slightly posterior to anterior end of soft anal fin.Pectoral fins small, extended latero-posteriorly, with their bases located mainly below upper margins of gill openings.No pelvic fins.Origin of exposed anal spines slightly posterior to anal.Caudal fin very small and rounded with 7-8 rays, confluent with soft dorsal-and anal-fins.First and second anal spines supported by well-developed first anal spine pterygiophore and third anal spine

Figure 6 .
Figure 6.Habitat at the type locality of Sinobdella longitubulus in the Datong-Jiang, a stream tributary to the Xun-Jiang of Zhu-Jiang Basin at Lilia Village, Pingnan County, Guangxi Province, China.
Figure 7. Bayesian Inference tree based on the COI gene for two species of Sinobdella and three species of Mastacembelus.Bayesian posterior probabilities (> 0.5), maximum Likelihood bootstrap values (> 50%) are shown, respectively.Dashes represent nodes with bootstrap support lower than 50%.

Table 2 .
Simon is not the collector as spiny eels are not found in Chinese fish specimens, available in the MNHN collection, caught by him.Information on specimens of Chinese spiny eels available in MNHN.