﻿Triplophysadaryoae, a new nemacheilid loach species (Teleostei, Nemacheilidae) from the Syr Darya River basin, Central Asia

﻿Abstract Triplophysadaryoae, new species, is described from the Sokh River, a former tributary of Syr Darya that today fails to reach the river, in the Sokh District, an exclave of Uzbekistan, surrounded by Kyrgyzstan. Triplophysadaryoae is distinguished from other species of Triplophysa in Central Asia by a truncate caudal fin with 13 or 14 branched rays, body without obvious mottling, dorsal-fin origin opposite to pelvic-fin insertion, and absence of the posterior chamber of the air bladder. Molecular data suggest that Triplophysadaryoae is closely related to T.ferganaensis from the Shakhimardan stream, a small tributary of Syr Darya in the Yordon village, another exclave of Uzbekistan in Kyrgyzstan. The two species were separated by a Kimura 2-parameter genetic distance of 2.8% in the mitochondrial DNA cytochrome c oxidase subunit I barcode region; they are also distinguished morphologically. A key to the species of Triplophysa in the Syr Darya basin and adjacent regions is provided.


Introduction
The genus Triplophysa Rendahl, 1933, comprises approximately 160 species (Fricke et al. 2022). The genus is widespread in western and central Asian waters, inland drainages of Balochistan, northwest to western Mongolia, and from the Qinghai-Tibet Plateau to the Yunnan-Guizhou Plateau in China (Zhu 1989;Wu and Wu 1992;Prokofiev 2017).
Syr Darya is the longest river in Central Asia and the second largest in volume after Amu Darya. It originates in the Fergana Valley at the confluence of the Naryn and Kara Darya, which flow from the Tian Shan Mountains and drain into the Aral Sea after passing through Uzbekistan, Tajikistan, and Kazakhstan. To date, eight species of Triplophysa species have been reported from the Syr Darya basin (Berg 1949;Turdakov 1963;Mitrofanov 1989;Sheraliev and Peng 2021a, b). The Fergana Valley is a mountainous region and its fish fauna differs from that of other regions in Uzbekistan. Triplophysa dorsalis (Kessler, 1872), T. elegans (Kessler, 1874), T. ferganaensis Peng, 2021, andT. strauchii (Kessler, 1874) have been recorded from the rivers of the Fergana Valley (Turdakov 1963;Sheraliev and Peng 2021b). The occurrence of T. stolickai (Steindachner, 1866) in the Fergana Valley is controversial and requires additional in-depth taxonomic research (Sheraliev and Peng 2021b;Sheraliev and Kayumova 2022).
The Sokh River is a tributary of the Syr Darya. It flows through the Sokh exclave of Uzbekistan, which is surrounded by Kyrgyzstan, and enters the Fergana Region. At present, the river fails to reach Syr Darya because its water is used for irrigation. The ichthyofauna of the Sokh River is almost unexplored. Here we report a new species of loach from the Sokh River.

Specimen sampling, preservation, and morphological analysis
Handling of specimens was consistent with the Republic of Uzbekistan Animal Welfare Laws (No. 545-I 26.12.1997; https://lex.uz/docs/-31719), guidelines, and policies approved by the Southwest University Local Ethics Committee for Animal Experiments. After euthanasia, specimens were fixed in 10% formalin and stored in 70% ethanol. The right-side pectoral fin was preserved in 95% ethanol for molecular analysis. Counts and measurements were performed following the procedures of Kottelat and Freyhof (2007) and, whenever possible, on the left side of the specimen. Measurements to the nearest 0.01 mm were acquired using digital calipers and performed point-topoint rather than based on projections. The nomenclature of the head pores follows Kottelat (1984). Standard length was measured from the tip of the upper jaw to the end of the hypural complex, whereas the length of the caudal peduncle was measured from the base of the last ray of the anal fin to the end of the hypural complex at the mid-height of the base of the caudal fin. The last two branched rays in the dorsal and

Phylogenetic reconstruction
The 652 bp COI gene sequence was used for phylogenetic analysis. Molecular analysis was conducted using three new COI sequences (T. daryoae, OK377300;T. elegans, OK377301;and T. uranoscopus (Kessler, 1872), OK377302), as well as 29 previously published sequences retrieved from the National Center for Biotechnology Information (NCBI) GenBank (Table 1) database (https://www.ncbi.nlm.nih.gov). The COI sequences were aligned using the Clustal_W algorithm in MEGA7 (Kumar et al. 2016), with manual checks for inconsistencies. The distances between different groups were determined using MEGA7, with 1000 bootstrap replicates calculated using the best-selected K2P model. For phylogenetic reconstruction, the datasets were analyzed based on Bayesian inference (BI) using MrBayes ver.3.2 (Ronquist et al. 2012) and maximum likelihood (ML) using MEGA7. MrBayes was run with six substitution Diagnosis. Triplophysa daryoae is distinguished from congeners by a combination of characters. It is distinguished from T. ferganaensis by possessing a truncate caudal fin with 13-14 branched rays (vs emarginate, 16 rays), 9 pores in the pre-opercular mandibula (vs 7-8), and a slenderer body (body depth at dorsal-fin origin 1.4-1.8 times the HL vs 1.2-1.4). It is distinguished from T. strauchii by absence of the posterior chamber of the air bladder (vs developed, with a long tube), possessing 9-10 inner gill rakers on the first gill arch (vs 12-16), and no obvious skin mottling (vs mottling). Triplophysa daryoae is also distinguished from T. dorsalis, T. dorsonotata, and T. elegans by having a truncate caudal fin (vs emarginate) and lacking a posterior chamber of the air bladder (vs developed in T. dorsalis and T. elegans). It is distinguished from T. sewerzowi, T. tenuis, and T. ulacholica by the dorsal-fin origin opposite to the pelvicfin insertion (vs anterior to vertical line of pelvic fin origin).
Body smooth and scaleless; cephalic lateral-line system well developed. Infraorbital and supraorbital canals stretching from the outer rostral barbel base and ethmoid, respectively, uniting in the posterior orbital region and extending posteriorly before converging with the supratemporal canal on the back of the head, and uniting with the lateral canal. Complete lateral line ending at caudal-fin base. Intestine moderately long, with two coils. Stomach U-shaped. Posterior chamber of the air bladder degenerated.
Coloration. Dorsal profile grayish-brown to pale green without regular blotches in live individuals, and dark gray-brown in preserved specimens. Ventral side of the body ivory with gray tint. Dorsal side of head with small irregular dark melanophores; dorsal side of caudal peduncle with four or five irregular dark brown blotches. All fin membranes hyaline and light gray, without obvious mottling (Figs 1, 3).
Sexual dimorphism. Mature males presenting granular tubercles on each side of the preorbital region and broadened and thickened external branched pectoral-fin rays dorsally covered by small and condensed epidermal breeding tubercles. Females without tubercles on the head and pectoral-fin rays.
Distribution and habitat. Triplophysa daryoae sp. nov. is known only from its type locality, the Sokh River, which originates in the Alay mountains and Turkestan range ( Fig. 4). Presently, Sokh River water is primarily used for irrigation and does not reach Syr Darya. The river is located at an altitude of 700-1500 m and is constantly flowing rapidly; the water is clear and cold (the water temperature was 7.3 °C when the holotype was caught), and the bottom consists of gravel and stone (Fig. 5). Triplophysa daryoae cohabited with Cottus spinulosus Kessler, 1872 and Schizothorax eurystomus Kessler, 1872, which are high-altitude fish species.
Etymology. Triplophysa daryoae is dedicated to Daryo Sheralieva, the lovely daughter of the first author. The specific name is a noun in the genitive case.

Molecular analysis
COI sequence data (Fig. 6) showed that Triplopysa daryoae belongs to a group of species with a wide distribution in the Syr Darya, Tarim, and Ili-Balkhash river drainages, an endorheic basin in Central Asia. This group is defined here as the T. dorsalis species group, and our molecular data suggest that it includes T. chondrostoma (Herzenstein, 1888), T. dorsalis, T. dorsonotata, T. elegans, T. ferganaensis, T. sewerzowi, T. strauchii, T. tenuis (Day, 1877), and T. ulacholica. The minimum K2P distances between T. daryoae and its closest relatives T. ferganaensis and T. tenuis were 2.8% and 4.5%, respectively (Table 3). Triplophysa daryoae was distinguished from its most closely related congener, T. ferganaensis, by 18 unique and diagnostic nucleotide substitution sites in the COI barcode region (652 bp) ( Table 4).
* The nucleotide position number was provided relative to the first nucleotide base of the complete COI gene of T. tenuis (KT224363).

Discussion
Triplophysa differs from other Nemacheilidae genera by presenting sexual dimorphism (Zhu 1989). The presence of specific aggregations of breeding tubercles on the dorsal surfaces of the pectoral fin and from the lower edge of the eye to the base of the outer barbel in mature males can be regarded as an autapomorphy and is the unique diagnostic character of Triplophysa (Prokofiev 2010). These tubercles are present in T. daryoae males. This study added three species (T. daryoae, T. elegans, and T. uranoscopus) to the previously published molecular reconstructions (Wang et al. 2016;Feng et al. 2019a;Wu et al. 2020). Our phylogenetic analysis was consistent with the results of previous molecular and morphological studies. Overemphasis on some characteristics with eco-phenotypic variation, such as body color, color patterns, barbel length, and mouth structure, when identifying Triplophysa species may be misleading (Ren et al. 2018). Therefore, it is advisable to employ a combination of morphological and molecular approaches to distinguish between loach species (Chen et al. 2021;Sheraliev and Peng 2021b;Deng et al. 2022;Lu et al. 2022).
Triplophysa dorsalis is recorded from the middle and upper reaches of Kara Darya, whereas T. elegans is only recorded from the upper reaches (Baltabaev 1971). Triplophysa strauchii occurs in all waters of the Fergana Valley, whereas T. ferganaensis has only been recorded from its type locality, the Shakhimardan stream (Sheraliev and Peng 2021b). These species are more similar to Triplophysa daryoae than to other congeners. Nevertheless, they can be easily distinguished from the new species based on morphology.
Triplophysa daryoae can be distinguished from T. ferganaensis, which is the most similar species in terms of morphometric characteristics and habitat conditions, using the following characteristics: caudal fin truncate with 13 or 14 branched rays (vs emarginated with 16 branched rays), 6 branched pelvic-fin rays (vs 7 or 8), 9-11 (modally 10) branched pectoral-fin rays (vs 11-13, modally 12); cephalic lateral-line system with 6 suborbital and 9 pre-operculo mandibular pores (vs 7 and 7 or 8, respectively), dorsal and caudal fins almost hyaline, and spots imperceptible (vs spots on dorsal and caudal fins clearly visible). Triplophysa daryoae is distinguished from T. strauchii, which is the most common species of Triplophysa in the waters of Fergana Valley, by the small number of vertebrae (4+35 vs 4+37-38); a smaller number of gill rakers in the inner row of the first gill arch (9 or 10 vs 12-16); absent posterior chamber of air bladder (vs developed, with a long tube); shorter body depth and width at dorsal-fin origin .4% of SL, respectively); dorsal-fin origin equal to pelvic-fin insertion (vs anterior to vertical line of pelvic fin); and upper and lower lobes of caudal fin equal (vs upper lobe slightly longer than lower lobe). Triplophysa daryoae differs from T. dorsalis and T. elegans, which are rare species in the Fergana Valley, according to the following characteristics: dorsal-fin origin equal to pelvic-fin insertion (vs anterior to vertical line of pelvic fin in both); 9 or 10 gill rakers in the inner row of the first gill arch (vs 13-16 in T. dorsalis); wider interorbital width (29.5-35.6% of HL vs 23.9-27.6% of HL in T. dorsalis); longer pectoral-pelvic distance (30.4-34.2% of SL vs 24.6-28.7% of SL in T. dorsalis); shorter dorsal-fin depth (14.9-18.5% of SL vs 19.8-22.8% of SL in T. dorsalis and 18.9-24.1% of SL in T. elegans); caudal peduncle equal to HL (vs usually shorter in T. dorsalis); caudal-peduncle depth 7.6-9.2% of SL (vs 6.6-7.1% of SL in T. elegans); posterior chamber of air bladder degenerated (vs developed in both); lips thick with furrows (vs smooth lips in T. dorsalis); and caudal-peduncle depth 2.2-2.9 times its length (vs 3.2-3.5 times in T. elegans). Moreover, the genetic distance between the Triplophysa daryoae T. dorsalis, T. elegans, T. ferganaensis, and T. strauchii (6.5%, 5.6%, 2.8%, and 6.8%, respectively), based on the mitochondrial COI barcoding region, is consistent with the species-level divergence in other fish taxa (Ward et al. 2005;Rosso et al. 2012;Abdulmalik-Labe and Quilang 2019;Freyhof et al. 2021).
Another ten species of Triplophysa occur in the Syr Darya basin and adjacent area of the Fergana Valley (Berg 1949;Turdakov 1963;Sheraliev and Peng 2021b). Among them, T. tenuis is similar to T. daryoae in its general body structure, especially in the caudal-fin shape. However, T. daryoae is distinguished from T. tenuis by presenting 9-10 gill rakers in the inner row of the first gill arch (vs 12-15); dorsal fin slightly posterior to the midpoint between the tip of the snout and caudal-fin origin (vs anterior to the midpoint between the tip of the snout and caudal-fin origin); caudal-peduncle depth 7.6-9.2% of SL (vs 5.2-5.7% of SL), head depth 52.1-60.6% of HL (vs 47.8-51.2% of HL); and caudal-peduncle depth 2.2-2.9 times its length (vs 4.1-4.3 times). Triplophysa daryoae is distinguished from T. paradoxa by the absence of scales (vs covered by scales), dorsal-fin origin opposite to pelvic-fin insertion (vs anterior to vertical line trough pelvic-fin origin), and dorsal-fin origin closer to the caudal-fin base than to the snout tip (vs closer to the snout tip). Triplophysa daryoae is distinguished from T. ulacholica by the shorter average caudal-peduncle length (20.8 ± 1.11% of SL vs 27.0 ± 0.24% of SL), deeper caudal peduncle (7.6-9.2% of SL vs 4.9-6.8% of SL), caudal-peduncle depth 2.2-2.9 times its length (vs 4.0-5.3 times), and smaller eye diameter (12.5-17.1% of HL vs 18.5-23.3% of HL). The new species can be distinguished from T. coniptera by the rounded edge of the pectoral fin (vs pointed); caudal fin truncate (vs deeply forked); snout length 7.4-10.0% of SL (vs 10.1-11.8% of SL); caudal-peduncle depth 7.6-9.2% of SL (vs 5.2-7.3% of SL); and caudal-peduncle depth 2.2-2.9 times its length (vs 3.4-4.5 times). Triplophysa daryoae is distinguished from T. sewerzowi by the following characteristics: dorsal-fin origin opposite to pelvic-fin insertion (vs anterior to vertical line of pelvic-fin origin), second branched ray of dorsal fin the longest (vs third or fourth), and degenerated posterior chamber of the air bladder (vs developed). It is also distinguished from T. dorsonotata by its smaller eye diameter (7.2 times of HL vs approximately 6 times), predorsal length longer than post-dorsal length (vs pre-dorsal length slightly shorter than post-dorsal length), cephalic lateral-line system with 6 suborbital pores (vs 7 or 8), and caudal fin truncate (vs emarginated). The new species can be distinguished from T. kungessana by its thick lips with furrows and papillae (vs almost thin and smooth, without clear fringes or papillae) and pelvic fin reaching the anus (vs not reaching the anus). Triplophysa daryoae can be distinguished from T. herzensteini by having 4+35 vertebrae (vs 4+39-41) and a caudal-peduncle depth 7.6-9.2% of SL (vs 5.9-6.8% of SL). Triplophysa daryoae can be distinguished from T. uranoscopus by its caudal-peduncle depth measuring 2.2-2.9 times its length (vs. 2.7-4.1 times); caudal-peduncle depth 7.6-9.2% of SL (vs 5.4-7.0% of SL); dorsal-fin origin opposite to pelvic-fin insertion (vs anterior to vertical line of pelvic-fin origin). It differs from T. lacusnigri by a shorter head length (4.3-5.0 times SL vs 3.4-4.5 times SL) and non-oblique head profile in front of the eye (vs strongly oblique); the tip of the pectoral fin is usually formed by 4 branched rays (vs 2 or 3).
Triplophysa dorsonotata, T. elegans, T. lacusnigri, T. tenuis, and T. uranoscopus from different water bodies in Central Asia have long been synonymized with T. stolickai due to their morphological resemblance (Berg 1949;Turdakov 1963;Zhu 1989;Wu 1992, Prokofiev 2010). We were unable to find T. stolickai in inland waters of Uzbekistan (Sheraliev and Peng 2021a). To confirm the existence of T. stolickai in the waters of Uzbekistan, further morphological and genetic taxonomic revisions of Triplophysa from this region, previously synonymized with T. stolickai, are required. However, Feng et al. (2019a) reported that T. stolickai from the Qinghai-Tibet Plateau represents an interesting case of morphological convergence and consists of distinct four lineages that are not closely related. In the phylogenetic tree presented here, T. stolickai nested in three lineages with genetic distances (K2P) from T. daryoae of 6.3%, 6.8%, and 9.7% (Fig. 6). Despite the high morphological diversity of T. stolickai, the new spe-cies differs from it by its caudal-peduncle depth (7.6-9.2% of SL vs 5.9-7.1% of SL in Ili River, 5.4-7.3% of SL in Tarim and Indus rivers, and 6.2-6.5% of SL in Yangtze River); interorbital width being 29.5-35.6% of HL ; head depth at nape 52.1-60.6% of HL (vs 46.1-51.5% of HL in Ili River); caudal-peduncle depth 2.2-2.9 times its length (vs 3.2-3.9 times in Ili River; 2.9-4.6 times in Tarim and Indus rivers; 3.6-3.8 times in Yangtze River); vertebrae 4+35 (vs 4+38-41); body without obvious mottling (vs. with mottling); supraorbital sensory canal always connected with the infraorbital canal (vs usually not connected); and 2 supratemporal pores (vs 3 or 4). Berg (1905:243) noted that the rivers of the Syr Darya and Tarim basins originate on a flat marshy plateau in the Tian Shan, where water streams periodically change their direction and is difficult to distinguish between waters in separate basins. On this basis, he explained the similarity of ichthyofauna in the highlands of Central Asia (Berg, 1905). Triplophysa daryoae is similar to some species of Triplophysa from the Tarim basin and adjacent regions, with normal eyes, scaleless body, body color, color patterns, and slightly laterally compressed caudal peduncle (Li et al. 2007;Cao and Zhang 2008). However, it can be distinguished from T. bombifrons, T. laterimaculata, T. moquensis, T. papillosolabiata, and T. zamegacephala by the posterior chamber of its air bladder being degenerated (vs developed); 9 or 10 gill rakers in the inner row of the first gill arch (vs 12-14, 12, 13-15, 12-15, and 15-19, respectively); dorsal fin inserted opposite to vertical through pelvic-fin origin (vs dorsal-fin origin anterior to pelvic-fin insertion); pelvic fin reaching the anus (vs not reaching in T. kaznakowi); pre-pelvic length 50.7-55.5% of SL (vs 56.3-60.5% of SL in T. laterimaculata); caudal-peduncle length 19.1-23.1% of SL (vs. 28.6-31.3% of SL in T. bombifrons and 23.3-29.4% of SL in T. papillosolabiata); caudal-peduncle depth 7.6-9.2% of SL (vs. 4.5-6.7% of SL in T. waisihani); and 4+35 vertebrae (vs 4+38-43 in T. moquensis and 4+39-41 in T. waisihani). T. daryoae can be distinguished from other Triplophysa species by the following characteristics: 39 vertebrae (vs 41-47 in T. orientalis); caudal-peduncle depth 2.2-2.9 times its length (vs 3.3 times in T. microphysa, and 6.7-8.7 times in T. incipiens); and posterior chamber of air bladder degenerate (vs developed in T. microphthalma).
A Triplophysa dorsalis species group is proposed here based on the molecular data and phylogenetic reconstruction obtained. The proposed species (see above) have also been nested in a single clade in previous phylogenetic studies of Triplophysa (Wang et al. 2016;Li et al. 2017;Feng et al. 2019a;Wu et al. 2020). However, no unique morphological synapomorphies that diagnose the T. dorsalis species group have been identified. In-depth morphological studies may clarify this issue in the future. Regarding biogeographical distribution, we hypothesize that species such as T. coniptera (Talas River), T. dorsonotata (Ili River), T. herzensteini (Ili River basin), T. kungessana (Künes River), T. paradoxa (Talas River basin) and T. salari (Chirchik River) also belong to this species group. In contrast, T. uranoscopus, which is widely distributed in the Zeravshan River, was not nested in this clade (Fig. 6). This result suggests that loaches from Amu Darya  Gill rakers in the inner row on the first gill arch 9-10; caudal peduncle depth 3 times less than its length .. analysis. We thank Davronbek Rustamov for preparing photos in Figs 1, 2. This work was funded by the grant from the National Natural Science Foundation of China (No. 32170457).