﻿A new species of torrent catfish, Liobagrusgeumgangensis (Teleostei, Siluriformes, Amblycipitidae), from Korea

﻿Abstract In a recent survey of populations of the Korean torrent catfish Liobagrus, a distinctive species was discovered from the Geum River and its tributaries flowing into the western coast of Korea, and here described as a new species, L.geumgangensissp. nov. It is distinguishable from other congeners by a combination of the following characters: I, 8 pectoral fin-rays; 52–56 caudal-fin rays; a relatively short occiput to dorsal-fin origin distance (6.9–9.8% SL); a short pelvic-fin insertion to anal-fin origin distance (11.9–17.3% SL); a long dorsal-fin base (10.6–13.5% SL); 8–9 gill rakers; 5–8 serrations on the pectoral fin; the body and fins are dark yellow, the margins of the dorsal, anal, and caudal fins are dark brown, but the outermost rim is faintly yellow. Analysis of the cytb gene also confirmed that L.geumgangensis is a monophyletic lineage distinct from other congeners.

Genus Liobagrus can be distinguished from the genus Amblyceps by its pale fin margins, adipose fin confluence with the caudal fins, rounded or truncated caudal fin (Wright and Ng 2008), and main distribution in eastern Asia.Including the most recently described species, L. brevispina, 14 species have been reported from the Yangtze River (Changjiang) in mainland China, five species from Korea, and one species from Japan, for a total of 20 species.Liobagrus from the Korean Peninsula have been described with the following characteristics: L. andersoni Regan, 1908c, is distinguished by upper and lower jaws of equal length; L. mediadiposalis Mori, 1936, by a shorter lower than upper jaw; L. obesus Son, Kim & Choo, 1987 Kim, 2010, by the broad yellowish, vertical, crescent-shaped band in the middle of the caudal fin; and L. hyeongsanensis Kim, Kim & Park, 2015 by its small size, with a maximum standard length of 90 mm, and body and fins that are entirely yellowish brown without distinct markings.
The Korean Peninsula is largely divided into western and eastern halves by the Baekdudaegan Mountain Range.Recent studies on freshwater fish from Korea have focused on taxonomic reviews of fish separated by this range: Koreocobitis rotundicaudata (West), Wakiya and Mori 1929 vs. K. naktongensis (East), Kim et al. 2000;Coreoleuciscus splendidus (West), Mori 1935 vs. C. aeruginos (East), Song and Bang 2015;Cobitis nalbanti (West), Vasil'eva et al. 2016 vs. C. hankugensis (East), Kim et al. 2003.Following this logic, it is suspected that subpopulations of L. mediadiposalis, which are widely distributed across river drainages from the western (Geum River) to the southeastern (Nakdong River) portions of South Korea, may actually represent distinct species (Son 1987;Kim et al. 2006;Kim 2013).In this study, we investigate the identity of L. mediaposalis from the Geum River and confirm its distinctiveness from L. mediadiposalis s. str., describing it here as Liobagrus geumgangensis, a new species.

Morphological analysis
All samples were collected using a scoop net, fixed in 10% formalin, and preserved in 80% ethanol.Count and measurement procedures were taken following Hubbs and Lagler (1964) and Wright and Ng (2008).Fin spines and soft rays were counted from radiographs and bone staining, and the last two elements of the dorsal and anal fins were counted as one ray.Vertebral counts were also made from soft X-ray radiographs (HA80, HITEX), and the three components associated with the Weberian apparatus were excluded (Chen and Lundberg 1995;Zhao et al. 2004).Gill raker counts were made on the left side on the upper limb of the first arch with an additional 20 specimens to avoid damage to the holotype and paratype specimens (see Comparative material examined).Caudal fin-ray counts include principal caudal-fin rays and dorsal and ventral procurrent caudal-fin rays.Measurements were taken using digital calipers (0.01 mm) and expressed as standard length (SL) percentages in Table 1 which included data, followed by the number observed for each count and measurements in parenthesis.All Korean specimens are deposited in the Faculty of Biology at Jeonbuk National University, Jeonju, Korea (CNUC herein; current acronym is JNUC).Other specimens were used as those deposited in the following institutions: Institute of Zoology, Chinese Academy of Sciences (CAS); Institute of Hydrobiology, Chinese Academy of Sciences (IHB); National Tsing Hua University (NTHUB).Counts and measurements for all paratypes, if different from the holotype, are presented in parentheses after the value for the holotype.

DNA analysis
For genomic DNA analysis, a piece of pectoral fin was dissected from the specimen and stored in 100% ethyl alcohol.Next, total DNA was extracted with the genomic DNA Prep Kit for blood and tissue (QUIAGEN Co., USA).The mitochondrial cytochrome b gene was amplified by PCR using the method of Kim et al (2006): forward (F-5' ACCACCGTTGTHNTTCAACTA 3') and reverse primer (R-5' TAGAATYYTRGCTTTGGGAG 3').PCR reactions were completed in a total volume of 50 µl, consisting of 2 µl genomic DNA, 1 µl of the 10 µM forward and reverse primer solutions, 24 µl of Premix Taq (Takara, Japan), and 22 µl of distilled water (Takara, Japan).PCR for all specimens was executed in GeneAtlas G-02 thermocycler (Astec, Japan) with initial denaturing step at 94 °C for 2 min and 30 cycles of 45 s at 94 °C, 1 min at 52 °C, and 1 min at 72 °C.A final extension step at 72 °C for 5 min.The PCR amplicons were visualized on a 2% agarose gel stained with LoadingStar (Dyne, Korea) together with negative controls and Takara 1 kB molecular size ladder for preliminary size determination.The final PCR products were run on an ABI-3730XL sequencer (Applied Biosystems, USA).
Diagnosis.Liobagrus geumgangensis can be distinguished from other species in the genus by the length of the upper and lower jaws, and the presence or absence of serrations on the posterior edge of the pectoral fin, which are representative characteristics of the genus Liobagrus.Liobagrus geumgangensis has a longer upper than lower jaw and serrations on the posterior edge of the pectoral fin.Species that share these characteristics with L. geumgangensis are as follows: L. mediadiposalis, L. somjinensis, L. hyeongsanensis, L. huaiheensis and L. pseudostyani.This new species can be distinguished by the relatively large number of serrations (5-8) on the posterior edge of the pectoral fin (vs.4-6 in L. mediadiposalis and L. somjinensis, 2-3 in L. hyeongsanensis, L. huaiheensis and L. pseudostyani).It can be further differentiated from L. huaiheensis and L. pseudostyani by the subtruncate caudal fin (vs.rounded).Liobagrus geumgangensis is distinguished from its geographically closest congeners L. mediadiposalis and L. somjinensis by the following combination of characteristics: the body and fins are dark yellow, and the margins of the dorsal, anal, and caudal fins are dark brown, but the outermost rim is faintly yellow (vs.broad yellowish outer margin of the fins in L. mediadiposalis and crescent-shaped band in the middle of the caudal fin in L. somjinensis); pectoral fin rays I, 8 (vs.both I, 7); caudal fin rays 52-56 (vs.both 57-61); a relatively short occiput to dorsal-fin origin (6.9-9.8%SL vs. 10.3-13.3% in L. mediadiposalis and 9.7-13.0 in L. somjinensis); a short pelvic-fin origin to anal-fin origin (11.9-17.3%SL vs. 15.3-20.9% in L. mediadiposalis and 13.3-18.7% in L. somjinensis); a long dorsal-fin base (10.6-13.5% SL vs. 7.7-10.4% in L. mediadiposalis and 8.8-11.3% in L. somjinensis); and 8-9 gill rakes (vs.7-11 in L. mediadiposalis and 7-9 in L. somjinensis) (Figs 1, 2).
Mouth terminal; lips thickened and papillate, premaxillary and mandibular toothpads curved, teeth small and ciliform or setiform, with upper jaw slightly longer than lower jaw.Four pairs of barbels; maxillary barbel longest, reaching to base of pectoral-fin spine; nasal barbel short, not reaching to posterior margin of preoperculum; outer mental barbel long, reaching to posterior edge of pectoral-fin base; inner-mental barbel shortest among four pairs of barbels, about half length of outer mental barbel, reaching to gill membrane.Body compressed and round, head depressed and caudal peduncle strongly compressed; dorsal and ventral profiles straight.Predorsal profile slightly sloping ventrally from dorsal fin to occiput.Eye smaller, dorsolateral, subcutaneous, ovoid.Snout rounded in dorsal view.Anterior nostril tubular, rim with a fleshy flap forming a short tube; posterior nostril porelike, rim posteriorly confluent with base of nasal barbel.Gill membranes narrowly joined at isthmus.Lateral line absent or vestigial.
Coloration.See Fig. 1 for general appearance.In life (Fig. 1): body generally brownish yellow, fading to light yellow on ventral surface, without any distinct markings.All barbels pale gray.All fins with similar color to body, without deep yellow outer margins.
Sexual dimorphism.The adductor mandibulae in males is somewhat swollen during the spawning season, from late April to June.
Etymology.Named after the Geum River (Geumgang in Korean), the type locality.We propose the Korean name Geumgang-Jagasari for this species.
Distribution.Liobagrus geumgangensis sp.nov. is restricted to some rivers flowing to the west coast of South Korea: Geum River and upper stream of Mangyeong River (Fig. 5).
Biology and habitat.Liobagrus geumgangensis is nocturnal and benthic.They inhabit the bottom stratum of large and small stone or pebble substratum in shallow waters, about 0.3-0.6 m deep, with running waters with moderately fast currents.The spawning season is from late April to June.The adult females reach up to 99.8 mm SL and lay eggs 2.5-3.0 mm (2.8±0.1) in diameter.The adult males guard their fertilized egg.They feed mostly on aquatic insect larvae such as those of the Trichoptera, Ephemeroptera and Diptera.

Molecular analysis
As a result of the cytb gene analysis, we confirm that samples from Liobagrus geumgangensis form an independent group distinct from the closely related species L. mediadiposalis and L. somjinensis.Kimura's 2-parameter distance analysis revealed genetic distances ranging from 0.2% to 2.3% within groups of L. geumgangensis populations.We found the genetic distance between L. geumgangensis and L. somjinensis to be 4.7% to 5.0%, which was smaller compared to that observed with L. mediadiposalis (5.8% to 6.1%; Fig. 4).These findings provide robust evidence that L. geumgangensis is rather genetically close to L. somjinensis, while suggesting that it is a distinctly different species from congeneric species.as L. geumgangensis, along with L. mediadiposalis, L. somjinensis and L. hyeongsanensis, have a lower jaw shorter than its upper jaw.Liobagrus mediaposalis, L. somjinensis and L. hyeongsanensis all have I, 7 pectoral-fin rays; just two species, L. andersoni and L. geumgangensis have I, 8 pectoral-fin rays.The pectoral fin rays are not used as a common diagnostic characteristic and are considered to be species-specific variables, but in contrast, some studies have suggested fixed results (Xie et al. 2022).Since this study did not measure a large number (N = 30) of L. andersoni specimens, it is possible that the number of pectoral fin rays may vary, but this study intends to use this as a comparative characteristic.
To summarize, within the Liobagrus distribution in Korea, it is noteworthy that L. geumgangensis is the only species with an upper jaw longer than the lower jaw and I, 8 pectoral fin rays.In addition, L. geumgangensis differs from L. somjinensis in several characters: absence vs. presence of a broad vertical yellowish crescent-shaped band on the caudal fin, with a deep black outer margin; 5-8 vs. 4-6 serrations on the pectoral fin; and 52-56 caudal fin rays vs. 57-61 (Fig. 4).
Liobagrus geumgangensis is allopatrically distributed in restricted river systems, the Geum River and a patch of the Mangyeong River, which flow toward the western coast of Korea.Unlike other Liobagrus species, L. somjinensis is restricted to rivers flowing to the west coast and Geogeum Island, the Somjin, Dongin and Tamjin rivers.Liobagrus hyeongsanensis has a much smaller distribution including the Hyeongsan River flowing into the east coast, whereas L. mediadiposalis is found only in the Nakdong River on the west coast (Fig. 5).Liobagrus andersoni is restricted to the Han River system alone.Meanwhile, L. obesus has a wide distribution including the western river systems of Geum, Yeongsan and Mangyeong, and their distribution overlaps with other congeners.Interestingly, based on morphological and genetic research, all six species of the Korean Liobagrus are endemic (Kim et al. 2006(Kim et al. , 2015;;Park and Kim 2010;Kim 2013).
Our results are consistent with a recent study by Kim et al. (2006) on the evolutionary relationships among L. mediadiposalis populations in Korea using cytochrome b sequences.Kim et al. (2006) considered Type I to be the Somjin River group, which included four populations (Dongin, Somjin, and Yeongsan rivers and Geogeum Island), and Type II as the Geum River group comprising a single population.Although they confirmed that the Geum River group was genetically distinct from L. mediadiposalis, the taxonomy of this population was not further investigated.Here, our analysis of seven different populations confirms that the Geum River group represents a distinct, unnamed species (Fig. 3).
The geographical isolation of the freshwater fish of South Korea is likely related to the geological origins of the Korean Peninsula.The Korean Peninsula was formed by active orogeny movements from the early Triassic to the Jurassic.The structure of the current complex topography is due to the tectonic shift concentrated in the Tertiary Period of the Cenozoic Era.As a result, Korea has an approximately 1400 km long mountainous region stretching from North Korea to South Korea.The peninsula, called the Baekdudaegan, contains at least 12 mountain ranges that have formed unique rivers, including the Han, Geum, Nakdong, Hyeongsan, and Mangyeong rivers (Chough et al. 2000;Sohn et al. 2001).Consequently, some Korean freshwater fish are distributed allopatrically, causing speciation.
According to Mori (1936) and Kim (1997), the freshwater fish fauna on the Korean Peninsula is divided into three biogeographical regions: northeast, west, and southern Korea subdistricts.Such biogeographical distributions based on large mountain ranges creating physical barriers and allopatric distributions lead to endemic species (Fig. 5).In particular, Korean cobitid fishes, including 10 endemic species, are the best example of a diverse regional distribution (Kim and Park 2002;Chae et al. 2019).Some mountain ranges between the west and south subdistricts serve as strong long-term barriers to recurrent gene flow.Such barriers led to the split of Coreoleuciscus splendidus populations into two genetically discrete subdistrict populations (Kim et al. 2012).Geographical speciation has also been observed among some species of the Cyprinidae family on the Korean Peninsula (Kim 1997;Kim and Park 2002;Park and Kim 2010).Isolation of fish populations in different river systems has led to high levels of endemism in Korean Liobagrus, including L. geumgangensis.

Key to species of Liobagrus distributed in Korea
1 The length of both jaws is the same, and the mouth is in front of the snout ... 2 -The length of both jaws is different, the lower jaw is shorter than the upper jaw, and the mouth is located below the snout ..

Figure 3 .
Figure 3. Phylogenetic tree using maximum likelihood (ML) method based on mitochondrial cytochrome b gene sequences of Siluriformes species including L. geumgangensis.Bootstrap support values based on 1000 replicates are displayed on each node as >50.The blue color of the branches indicates L. geumgangensis.

Table 2 .
List of mitochondrial cytb sequences newly obtained in this study and downloaded from GenBank with information on collection sites in South Korea and country of origin.