Zoosyst. Evol. 97 (2) 2021, 471-482 | DOI 10.3897/zse.97.72181 yee BERLIN Contribution to the trout of Euphrates River, with description of a new species, and range extension of Salmo munzuricus (Salmoniformes, Salmonidae) Davut Turan!, Ismail Aksu!, Miinevver Oral', Ciineyt Kaya, Esra Baycelebi! 1 Faculty of Fisheries, Recep Tayyip Erdogan University, 53100 Rize, Turkey http://zoobank.org/D06D2FA 3-C8F9-4799-9E39-3 E3ACA5A8337 Corresponding author: Ismail Aksu (ismailaksu@erdogan.edu.tr) Academic editor: Nicolas Hubert # Received 26 July 2021 # Accepted 15 September 2021 # Published 18 October 2021 Abstract In an effort to reveal the Euphrates trout taxonomy, the Karasu River, which is one of the eastern drainages of the river, was investi- gated and three independent populations were identified. Result revealed that two populations belonged to Salmo munzuricus, which was known only in Munzur River, while the other population belonged to an unnamed species. Salmo baliki, a new species, is de- scribed from the Murat River, a drainage of Euphrates River. It differs from Sa/mo species in adjacent water by the combination of the following characters: a grayish body; commonly one, rarely two pale black spots behind eye and on cheek; two to seven black spots on opercle; a few black spots on back and upper part of flank, missing on predorsal area; few to numerous large irregular-shaped red spots in median, upper and lower part of flank, surrounded by a large irregular-shaped white ring; the number of black and red spots not increasing in parallel with size; maxilla short and narrow; adipose-fin medium size, no or rarely one or two red spot its posterior edge; 107-118 lateral line scales; 24—28 scales rows between dorsal-in origin and lateral line; 18—22 scale rows between lateral line and anal-fin origin; maxilla length 7.7—9.1% SL in males, 8.2—9.6 in females. Finally, the genetic study of the Cyt 6 mitochondrial gene confirmed the morphological data, suggesting the separation of S. baliki from other Salmo species. Key Words Anatolia, cytochrome b, freshwater fish, Salmo, taxonomy Introduction Anatolia has a high level of species richness and ende- mism, thus it has been classified as a European biodiver- sity “hot-spot” (Kosswig 1955; Sekercioglu et al. 2011), which has also positively reflected in salmonid biodiversi- ty (Bardak¢1 et al. 2006). Salmo trutta L. 1758 is the most widely distributed freshwater fish native to the Palearctic region. Its natural habitat extends from Northeast Rus- sia and Norway, southward to the Atlas Mountains, also, from the spring waters of the Aral Sea to Iceland (Bernat- chez 2001; Lobon-Cervia 2018 and references there in). Initially, all Anatolian trout had been grouped within the S. trutta or tts subspecies (e.g. Kuru 1975; Geldiay and Balik 2007). Further studies based on morphology (Tur- an et al. 2012, 2014a, 2014b, 2017; Turan and Baycelebi 2020) and genetic-aided morphology (Turan et al. 2010, 2011, 2020) of Anatolian trout have revealed a much more complex species structure. Overall, fourteen species have been identified in Anatolia within the last decade. Based on our current knowledge, the upper Euphrates River is one of the most species-rich areas for the ge- nus Salmo genus including four well-described species: Salmo euphrataeus Turan, Kottelat & Engin, 2014 from the streams Senyurt, Kuzgun, Rizekent, Agircik and Sirh, northern Euphrates; S. okumusi Turan, Kottelat & Engin, 2014 from the streams Géksu, Gokpinar and Stirgu, west- ern Euphrates; S. munzuricus Turan, Kottelat & Kaya, Copyright Turan D et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 472 Black Sea Ze ae it _ gvaninen. uphrates River Turan, D. et al.: Contribution to the Euphrates trout o Karasu River AA r Murat River SS ene : )— Murat River “a Lae — @ ma aay \ 7, 5 Euphrates River @ Salmo munzuricus HE Salmo okumusi * Salmo baliki ¢ Salmo euphrataeus A Salo fahrettini Figure 1. Distribution of Sa/mo species in the Euphrates River basin. 2017 from the stream Munzur, northwestern Euphrates; S. fahrettini Turan, Kalayci, Bektas, Kaya & Baycelebi, 2020 from the streams Omertepe suyu and Tekke, north- ern Euphrates. Traditionally, five major evolutionary lineages of brown trout were described based on their origin, and phylogenetic; including the AD (Adriatic origin), AT (Atlantic), DA (Danubian), MA (Marmaratus) and ME (Mediterranean) (Bernatchez 2001). Further investiga- tions identified new lineages as Duero from Spain (DU; Suarez et al. 2001), TI from Turkey (Tigris; SuSnik et al. 2005; Bardakc¢i et al. 2006), Dades from Morocco (Snoj et al. 2011) and from Northern Africa (Tougard et al. 2018). Additional molecular studies have placed the trout species from the Euphrates River drainages in the Danu- bian (S. euphrataeus and S. fahrettini), and the Adriatic (S. okumusi and S. munzuricus) lineages providing the significant species diversity in the Euphrates. In the scope of this study, three additional trout pop- ulations in the Murat River were determined. To reveal the taxonomic status of these novel populations, morpho- logic and molecular studies were carried out to compare them with the previously identified species in the adya- cent waters. Our studies demonstrated that two of these populations belonged to the S. munzuricus, which was previously known from a single locality, while the oth- zse.pensoft.net er population belongs to an unnamed species within the Adriatic lineage. Material and methods The field work was carried out by following the guide- lines of the Local Ethics Committee of RTE University for the use of animals in scientific experiments with a per- mit reference number of 2014/72. Samples were collect- ed from the stream Sinek, drainage of the Murat River, A&r1, and eastern Turkey (Figure 1). This water is known to be one of the uppermost tributaries of the Euphrates River. Samples were caught using an electrofishing de- vice (Samus, 1000). First, live photographs were taken in an aquarium, filled with the water of the sampling res- ervoir so as to capture the natural coloration and patterns of the specimens. Then, anesthesia was performed using tricaine methane sulphonate solution (MS222). Subse- quently, fin clips were collected from one of the pelvic fins, placed into 96% ethanol, for molecular work. Fol- lowing a surgical procedure, samples were fixed in 4% formaldehyde in the vertical position. These specimens were taken to FFR, Zoology Museum of the Faculty of Fisheries, Recep Tayyip Erdogan University, Rize for de- tailed morphologic analysis. Zoosyst. Evol. 97 (2) 2021, 471-482 Morphological analyses Turan et al. (2010) was used as a guideline for morpho- metric analysis. All measurements were carried out in the form of point to point approach (projections were re- fused) using a dial caliper calibrated to 1 mm. Specific to the present study, the last two branched rays articu- lating on a single pterygiophore in the anal and dorsal fins were counted as “1/2”. Comparative materials used in this study were listed in Turan et al. (2010, 2011, 2012, 2014a, 2017, 2020). DNA extraction, PCR and sequencing Total genomic DNA was extracted from the ethanol-fixed fin clips using DNeasy Blood & Tissue Kit (Qiagen, USA) following the manufacturer’s protocol carried out in the Qiacube Automated DNA purification system. The DNA concentration and purity of each sample were assessed by spectrophotometry (Nanodrop, 2000/c, Thermo Scientific, USA), while the integrity was assessed by 0.8% agarose gel electrophoresis. Mitochondrial cytochrome b (Cyt 5b) gene was amplified using SsaL14437 (Warheit and Bow- man 2008) and StrCBR (Turan et al. 2010) primer pairs fol- lowing the PCR conditions specified in Turan et al. (2020). The amplicons were visualized on UV Quantum-—Capt ST4 system (Vilber Lourmat, France) and sequenced in both di- rections by Macrogen Inc. (Amsterdam, Netherlands). 473 Phylogenetic analysis A total of 65 Cyt b sequences were assessed from the Sal- mo species (Table 2) inhabited in the Tigris, Euphrates and Kura River drainages as well as the Black Sea and eastern Mediterranean Sea basins. Generated sequences were aligned using BioEdit 7.2.5 (Hall 1999) with Clust- al W (Thompson et al. 1994). Trimming was essential thus, applied to both ends of the fragments to set the equal lengths of 993 bp for each and every fragment. The phylogenetic relationships among Salmo species were assessed by maximum likelihood (ML) approach in MEGA X software (Kumar et al. 2018) and by Bayesian analysis (BI) in MrBayes v3.2.1 (Ronquist et al. 2012). The most appropriate evolution model of nucleotide substitution was selected by the Akaike Information Cri- teria (AIC) and Bayesian Information Criteria (BIC) ap- proaches in jmodelTest 0.1.1 (Posada 2008). ML tree was generated by selecting TrN+I+G (Tamura and Nei 1993) model according to AIC and 1000 bootstrap replicates ap- plied. The BI was generated according to the TrN+I+G (Tamura and Nei 1993) model that the evolution model was selected by the lowest BIC score. For BI, analyses were run for 110° generations with Metropolis coupled Monte Carlo Markov Chains (MCMC) sampled every 1000 generations. an outgroup so as to root the phylogenetic tree. The new sequences generated in the present study were deposited Figure 2. Salmo baliki, FFR 3242, holotype, 212 mm SL, male; Turkey: stream Sinek, a tributary of Murat River. zse.pensoft.net 474 Turan, D. et al.: Contribution to the Euphrates trout Figure 3. Salmo baliki, FFR 3234, paratypes, a. 216 mm SL, male; b. 170 mm SL, male; c. 164 mm SL, female; Turkey: stream Sinek, a tributary of Murat River. to ocak under the accession HDS Se NV 366844— MW 366860 and MW382946—-MW3 0 (Table 2). Results Salmo baliki sp. nov. vnhank argo /LOAR2BA1_25BA_ADAB_AACBRYOBRLORREQR2RNA LOO DALI 1k ore/69483E41-85FA-42AF \ACF-2ZE0IYBBESSBVUA F raise 2-4 Holotype. FFR 3242, 212 mm SL; Turkey: Agri Prov- ince: stream Sinek a tributary of Murat River at Taslicay, 39.758749°N, 43.464480°E. zse.pensoft.net Paratypes. FFR 3234, 6, 132-276 mm SL; same data as holotype. —FFR 3205, 3, 175-267 mm SL; Turkey: Aér1 Province: a tributary of Murat River 39.730705°N, 43.481869°E. Additional record. Turkey: Ari Province: stream Cuma at Cumacay, 39.919118°N, 43.192272°E. Diagnosis. Salmo baliki differs from the other species of trout recorded from the Euphrates and Tigris River drainages (S. euphrataeus, S. okumusi, S. munzuricus, S. fahrettini and S. tigridis) by having large and irreg- ular-shaped red spots on its body (red spots larger than pupil, vs. smaller than pupil). Salmo baliki further differs from S. euphrataeus by the general body color silvery in Zoosyst. Evol. 97 (2) 2021, 471-482 live (vs. brownish), a shorter head in the male (length 24— 27% SL, vs. 27-31), a shorter maxilla in the male (length 8—9% SL, vs. 10-11), and a shorter mouth gape (12-14% SL in male, 11—12 in female, vs.14—-17 in male, 12—15 in female). Salmo baliki further differs from S. munzuricus by having fewer black spots in postorbital and suborbital areas (commonly 1, rarely 2, vs. 3—24); fewer black spots on the body (less than 30, vs. more than 80 in adult spec- imens), whose number does not increase with size (vs. number increasing with size); the black spots circular (vs. irregularly shaped); commonly plain or rarely two round- ish red spots on posterior edge of the adipose-fin in male and female (vs. plain or the adipose-fin with a very narrow white margin, then a red submarginal band, then a white band or spots, then a red band again in males, Figures 3, 5), a smaller adipose-fin (8-9% SL in male, 7—8 in female, vs. 9-12 in male, 8—10 in female) a deeper anal-fin in females (16-18% SL, vs. 13-16), a greater anal-fin base (10-12% SL, vs. 8-10), a longer median caudal-fin rays (14-16% SL, vs. 11-14), a greater mouth gape in females (12-13% SL, vs. 10-12), a deeper maxilla in females (maximum maxilla depth 3-4% SL, vs. 2—3), a smaller distance be- tween the adipose and caudal fins in males (15—16% SL, vs. 16-18), and fewer lateral line scales (107-118, vs. 116-123). Salmo baliki further differs from S. okumusi by having one or two pale black spot in postorbital and sub- orbital area (vs. 1-22), fewer black spots on opercle (3-7, vs. 8-17), fewer black spots on the body in specimens smaller than 210 mm SL (less than 30, vs. more than 90), presence black spot on body in all size (vs. the body with black dots in specimens larger than about 230 mm SL), the shape of the black spots ocellated (vs. irregularly shaped), the black spots scatter on back and upper part of flank (vs. whole flank covered black spots or dots), no black spots on top head (vs. 5—18), one or two dark bands on posterior part of the flank (vs. four dark bands in specimens larger than about 230 mm SL), the parr marks vertically oblong (vs. vertically elongate), the shape of the black spots ocel- lated (vs. irregularly shaped), a more slender dorsal-fin in males (16-17% SL, vs. 17-19), a shorter pectoral-fin in males (17-19% SL, vs. 19-21), a smaller eye diame- ter (4—5% SL in males, 4—6 in females; vs. 5—7 in males, 6-8 in females), a shorter maxilla in males (8—9% SL, vs. 9-11) (see Figures 3, 6). Salmo baliki further differs from S. tigridis by having fewer scale rows between the dor- sal-fin origin and the lateral line (24—28, vs. 32-35); fewer scale rows between the end of the adipose-fin base and the lateral line (13-15, vs. 19-20), a slenderer caudal pedun- cle depth (11-12% SL, vs. 12-13). Salmo baliki further differs from S. fahrettini by having fewer black spots on its body (less than 30, vs. more than 80); the black spots scatter on back and upper part of flank (vs. scattered on back, middle and upper part of flank, and anterior part of lower half of flank), their number not increasing with size (vs. increasing with size), fewer red spots on body (fewer than 42 in adult specimens, vs. more than 70 in adult spec- imens), their number not increasing with size (vs. increas- ing with size). See Discussion for comparison with other trout in adjacent waters of Anatolia. 475 Table 1. Morphometry of Salmo baliki (holotype, FFR 3242; paratypes FFR 3205, n= 3 and FFR 3234, n =6). The calcula- tions include the holotype. Holotype Paratypes Sex male male Female Number of n=4 n=5 specimens Standard length 212 164-250 132-267 (mm) In percentage of standard length Head length 26.9 Predorsal length 47.4 Prepelvic length 52.7 Preanal length 72.7 Body depth at 23.9 dorsal-fin origin Body depth at 19.1 anal-fin origin Depth of caudal 10.6 Range(mean) SD Range(mean) SD 24.1-26.9 (25.7) 1.1 24.2-26.0 (25.2) 0.7 45.0-49.7 (47.7) 1.8 46.2-48.2 (47.4) 0.8 52.7-55.6 (54.3) 1.1 52.9-55.3(54.1) 1.0 72.7-76.0 (74.8) 1.3 72.6-77.7 (75.2) 2.0 23.6-26.4 (24.7) 1.2 22.2-25.8 (24.3) 1.4 18.5-20.8 (19.5) 1.2 17.4-20.1(18.9) 1.2 10.6-11.7 (11.0) 0.5 10.6-11.9(11.2) 0.5 peduncle Length of caudal 17.3. 16.1-17.9(17.2) 0.7 15.8-17.9(17.0) 0.9 peduncle Distance 16.00 15.4-16.4(15.8) 0.4 16.1-17.9(16.6) 0.7 between adipose- and caudal-fins Body width at 11.9 9.6-11.9(10.6) 0.9 8.9-11.7(10.5) 1.1 anal-fin origin Length of 12.2 9 12.2-14.8(13.5) 1.0 12.9-14.0(13.5) 0.4 dorsal-fin base Depth of dorsal- 16.7. 16.0-17.2 (16.6) 0.5 12.8-18.2(15.9) 2.0 fin Length of 17.8 17.0-19.0(17.9) 0.8 17.2-20.3(19.1) 1.2 pectoral-fin Length of 4.2 4.0-5.3(4.7) 0.6 3.9-5.0(4.4) 04 adipose-fin base Depth of 8.4 7.5-8.7 (8.2) 0.5 7.1-8.3(7.7) 0.6 adipose-fin Length of pelvic: 14.5 13.8-15.8(14.8) 0.8 14.0-15.2(14.7) 0.5 fin Depth of anal-fin 16.9 15.8-18.0(16.9) 0.8 16.3-18.3(17.4) 0.8 Length of anal- 10.3. 10.3-11.3(10.9) 0.4 10.2-11.8(10.9) 0.6 fin base Length of upper 17.0 caudal-fin lobe Length of 14.3 median caudal- fin rays Length of lower 17.5 caudal-fin lobe 15.3-17.2 (16.3) 0.8 15.2-18.3(16.7) 1.1 13.5-14.7 (14.3) 0.4 13.5-15.7(14.2) 0.9 15.6-18.2 (17.0) 1.2 14.4-19.2(16.9) 1.9 Snout length 8.1 6.3-8.3(7.2) 09 6.6-7.6(7.1) 0.5 Distance 4.5 3.7-5.0(4.4) 05 4.0-4.7(4.3) 0.2 between nasal openings Eye diameter 4.0 3.64.9(4.4) 05 3.6-5.8(5.1) 0.9 Interorbital 79 7.1-9.3(8.3) O08 6.9-8.1(7.5) 0.5 width Head depth 12.3 11.2-13.4 (12.5) 0.9 11.4-13.5(12.5) 0.9 through eye Head depth at 16.6 nape Length of 7.7 maxilla Maximum height all of maxilla Width of mouth 9.4 gape Lengthof mouth 13.9 gape 15.0-17.7 (16.3) 1.0 16.0-18.6(16.8) 1.0 7.7-9.1(84) 0.5 8.2-9.6(8.7) 0.6 2.5-3.1(2.9) 0.3 2.6-3.9(3.2) 0.5 8.6-10.5(9.6) 0.7 8.7-10.1(99.1) 0.6 11.7-13.9 (12.8) 0.9 11.6-12.6(11.8) 0.3 Description. The general appearance is shown in Fig- ures 2-4, morphometric data are in Table 1. Body deep, compressed laterally, its depth approximately equal to head length. Dorsal profile markedly arched and ventral profile less arched than the dorsal profile. Head short, upper profile zse.pensoft.net 476 Table 2. Materials used in genetic analysis. n” Species baliki munzuricus okumusi opimus chilo labecula platycephalus fahrettini coruhensis rizeensis euphrataeus Caspius tigridis trutta obtusirostris ohridanus Salar Sample 5 WNHNDNM WWWW WW WWW WwW ol Ww Locality Turkey: Agri, Sinek stream, Murat River, Euphrates River Turkey: Tunceli, Munzur Stream, Euphrates Turkey: Agri, Murat River, Euphrates River Turkey: Sivas, GOkpinar Stream, Euphrates Turkey: K.Maras, GOksun, Ceyhan River drainage Turkey: Sivas, Akdere stream, Ceyhan River drainage Turkey: Nigde, Ecemis stream, Seyhan River drainage Turkey: Kayseri, Pinarbasi stream, Seyhan River drainage Turkey: Erzurum, Omertepesuyu Stream Euphrates Turkey: Rize, Cayeli Kanlidere Stream Turkey: Rize, Kangel stream Turkey: Rize, Alakoz stream Turkey: Erzurum, Sirli Stream, Euphrates Turkey: Ardahan, Toros Stream, Kura River drainage Turkey: Ardahan, Derindere stream, Kura River drainage Turkey: Ardahan, Karaman stream, Kura River drainage Turkey: Van, Catak Stream Italy: Flumendosa France: Vidourle Slovenia: Volaja United Kingdom: Camel Austria: Kleiner Kamp Norway: Leksa Turkey: Van, Arpet Stream, Tigris Turkey: Bitlis, Sapur Stream, Lake Van Bosnia and Herzegovina: Neretva Macedonia: Lake Ohrid Norway: Ims MW382946- MW366853- MW366850- MW36684 7- MW366844— Accession number MW366856-— MN815914 MN815915 MN815913 MN815912 MN815910 MN815910 MN815911 MN815909 MN815909 MN815909 MN815916 LT617538 LT617535 LT617539 LT617540 KF985687 JX960836 MT981164-M7T981165 MT981168-MT981169 JX960841 AFO53590 JX960834 MW366860 MW382950 MW366855 MW366852 MW366849 MW366846 Turan, D. et al.: Contribution to the Euphrates trout Reference This study Turan et al. 2020 This study et al. 2020 is study is study is study This study Turan et al. 2020 Turan et al. 2020 Turan et al. 2020 Turan et al. 2020 Turan et al. 2020 Turan et al. 2020 Turan et al. 2020 Turan et al. 2020 Turan et al. 2020 Tougard et al. 2018 Tougard et al. 2018 Tougard et al. 2018 Tougard et al. 2018 Schenekar et al. 2014 Créte—Lafreniére et al. 2012 Kaya 2020 Kaya 2020 Créte—Lafreniére et al. 2012 SuSnik et al. 2006 Créte—Lafreniére et al. 2012 Tura h h Th hH Figure 4. Salmo baliki, FFR 3205, paratypes, a. 250 mm SL, male; b. 267 mm SL, female; Turkey: stream Sinek, a tributary of Murat River. straight both on the snout and above the eye in male, straight above the eye and convex on snout in female. Mouth small, terminal or slightly subterminal in male, subterminal in fe- male. Tip of lower jaw slightly curved upwards, pointed, zse.pensoft.net with a slightly-developed process at symphysis in male larger than 200 mm SL. Maxilla short, reaching slightly beyond posterior margin of the eye in males and female larger than about 200 mm SL. Snout somewhat long, with Zoosyst. Evol. 97 (2) 2021, 471482 477 Figure 5. Salmo munzuricus: a. FFR 3226, 211 mm SL, male; Turkey: Tunceli Prov., stream Kalan; b. FFR 3241, 205, male; Tur- key: Mus Prov., stream Mengel; ce. FFR 3226, 240, male; Turkey: Agri Prov., stream Alakoglu. pointed tip in male, rounded in female. Adipose fin some- what large, its height 7.5—8.7% SL in males and 7.1—8.3 in females. Largest observed specimen 250 mm SL. Dorsal fin with 3—4 unbranched and 8-10 branched rays, its distal margin slightly convex. Pectoral fin with 1 unbranched and 10-11 branched rays, its external mar- gin slightly convex. Pelvic fin with 1 unbranched and 7-8 branched rays, its external margin slightly convex. Anal fin with 3 unbranched and 7-9 branched rays, its distal margin convex anteriorly and straight or concave posteriorly. Caudal fin deeply emarginated in specimens less than 160 mm SL, slightly emarginated or truncate in specimens larger than about 200 mm SL, lobes slightly pointed. Lateral line with 107-118 scales; 24-28 scale rows between dorsal-fin origin and lateral line; 18-22 scale rows between anal-fin origin and lateral line; 13-15 scale rows between origin of the adipose fin and lateral line. Gill rakers 16—18 on first gill arch. Coloration. In formalin: General coloration of fresh- ly preserved specimens silvery on back and flank, yel- lowish on the belly. One pale black spot in postorbital and suborbital areas, greater than pupil; three to seven black spots on opercle, approximately smaller than pu- pil. Black spots on body few (fewer than 30), smaller than the pupil, ocellated, scattered on the upper part of flank (missing in back). No black spot on top of the head. Red spots few (fewer than 30), large (greater than pupil), irregularly-shaped, surrounded by an irregularly shaped narrow ring, organized in two to four irregular longitudinal rows on median part of the body, and half zse.pensoft.net 478 Turan, D. et al.: Contribution to the Euphrates trout Figure 7. a. Stream Sinek, Murat River, Turkey; type locality of Salmo baliki: b. Stream Alakoglu; c. Stream Mengel, Murat River Turkey, two new localities of S. munzuricus. of lower part of the flank. The number of black and red spots on flanks does not increase with increasing size. Dorsal fin grey, with three or four rows of black spots (smaller than pupil) and one or two rows of red spots (smaller than pupil). Caudal fin dark gray; pectoral, anal pelvic fins greyish. Adipose-fin plain grayish, rarely one or two red spots on its posterior edge. Eight to nine ob- long parr marks on the body, distinct 1n specimens up to about 190 mm SL. 1-2 vertical bands on posterior half of flank in most specimens. Distribution and habitat. Salmo baliki inhabits clear and moderately swift-flowing water, with a substrate of stones and pebbles. The observed material for this spe- zse.pensoft.net cies has only been collected from stream Sinek, drain- age of Murat River (Figures 1, 7a). The species has not been found in adjacent waters except stream Cumac¢ay (39.919118°N, 43.192272°E) that is located approx- imately 32 km northwest of Sinek, another drainage of Murat River. However, there was no opportunity to carry out survey in the stream Cumagay location. Following solid evidences of shape and size of the spots from the video records shown by local people, this location will further be investigated in the near future. Conservation status. There is serious pressure on the populations of Salmo baliki due to overfishing. The spe- cies is taken for curative purposes, hence demand is very Zoosyst. Evol. 97 (2) 2021, 471-482 Tree scale: 0.01 ’-———-“¥+] 97/1.0 479 axo6os34 §. salar AF0S3590 §. ohridanus Ix960841 § obfusirestris LT617535 LT617538 LT61/539 LT61/540 KF985687 JXS60836 MT981169 MT981168 MT981165 MT981164 ; MN&15916 100/10) g15916 - MN815916 MIN815913 7310.94) ing15913 9, MN815913 7510.66 MNB15912 MN815912 MN815912 - MN8i5910 MNS15910 MN815910 MN&15910 MN&15910- MN815910 MIN815911 MN815911 MNB15911 MNB815909 MNB15909 MNB15909 MNB15909 MN&15909 MNB15909 MIW366855 MW 366854 MW366853 - MW366852 MW366851 MW366850 MW366849 MV S66845 MW366847 MW366846 MW366845 MW366844 Mw'382950 MWw382949 MW382948 MW382947 MW2382946 MN815914 | MN815914 89/1.0 F MN@15914 MN815915. MNB15915 MNB15915 MW366860 MW366859 MW366858 MW366857 MW366856 60.9: 93/1.0 P4095 84/083 TT7/0.87 68/0.98 §9/1.0 64/094 S. trutta | S. tigridis S. fahrettini S. coruhensis S. rizeensis S. euphrataeus S. caspius | S. opimus | S. chilo S. labecula S. platycephalus S. MMM IMTICUS | S. okmmusi S. baltki Figure 8. Bayesian inference (BI) phylogenetic tree based on Cyt b sequences of Salmo species. ML and BI methods generated the similar topologies and therefore only the BI tree is shown. The bootstrap values of ML and posterior probability values of BI are indicated on nodes (ML/BI). high. Within the first fieldwork in the area which was carried out in 2006, in total 3 specimens were found in the middle of the stream (39.730705°N, 43.481869°E), however no specimens were detected in the same loca- tion during the recent survey, only a small population observed about 4 km upstream (a restricted area, far from the villages, and the only transportation 1s provid- ed through a rough and muddy road). Taking all these factors into account, endemic S. baliki is stuck in a very limited area, thought to be under a serious threat. There- zse.pensoft.net 480 fore, there is a need for the species to be conserved under international legislation. Sexual dimorphism. The snout of the male is more pointed than that of the female. The depth of the adi- pose-fin and the length of the mouth gape in male are greater than those of the female. Etymology. The species is named after Dr. Suleyman Balik (Turkey), taxonomist, in appreciation of his contri- butions to the freshwater fish fauna of Turkey. Phylogenetic placement of Salmo baliki. We analyzed a total of 65 sequences (22 new sequences in this study and 43 sequences from GenBank; Table 2) to assess if the phy- logenetic relationship among Salmo sp. Salmo baliki new Species is genetically different from the other Sa/mo species. The nodes separating the species in the phylogenetic tree to- pology of the Cyt 6 gene were supported by high posterior probability and bootstrap values. (Figure 8). The bootstrap values in ML analysis are relatively low compared to the posterior probability values in BI analysis. However, the two tree topologies do not contradict each other. According to the result of phylogenetic analysis, Salmo baliki is a sister taxon to S. munzuricus (Figure 8). For Salmo species, phy- logenetic tree topology corresponds exactly with the fiction formed as a result of morphological data. Range extension of Salmo munzuricus. Within the aim of the present study a geographic range extension for Salmo munzuricus was also recorded. This species was previously only described from Munzur River, north western Euphrates (Turan et al. 2017). Here, two new localities have been identified for S. munzuricus (Figure 7b, c). These new localities in the Murat River, located 140 and 340 km east of the previously known distribu- tion range of the species, reveal the scarce bio-geographic knowledge of the species in the Euphrates basin. Discussion The population of Salmo baliki has been experiencing a serious pressure caused by local people and fisherman. The trout inhabiting stream Sinek is thought to be a ‘healer fish’, as appeared in local and national press, thus sold for higher prices throughout the country. This is the main rea- son behind the relatively low number of specimens investi- gated in the present study as opposed to standard morpho- metric studies (10 fish versus 25 fish). However, evidence, discussions with local people and video recordings suggest an additional locality for S. baliki, soon to be confirmed. Trout inhabit cold, well-oxygenated waters where the flow 1s relatively high and species get restricted to such locations. This leads to ecological isolation from the oth- er populations inhabiting same water bodies. Hence, this, in turn, has a significant effect on speciation. Although there are significant morphological differences among the trout species, relatively lower genetic distances in mtD- NA sequences indicate an early stage of speciation taking place in Salmo genus within the course of evolution. In total, fifteen native trout species have been identified in zse.pensoft.net Turan, D. et al.: Contribution to the Euphrates trout Turkey, six of which are known from the Euphrates and Tigris drainages, namely; Salmo tigridis, S. okumusi, S. euphrataeus, S. munzuricus, S. fahrettini and S. baliki (in the present study). Those of Salmo baliki, S. munzuricus and S. okumusi belong to Adriatic linage. Molecular dis- tance among these species is not very distinct, however, the remarkable morphological differences easily separate these species which are presented above in the diagnosis section (see also Figure 2-4, 5 and 6). Salmo baliki is easily distinguished from S. platyceph- alus, S. chilo, S. labecula and S. opimus, all from streams draining to the Mediterranean, by zero to two dark bands on the posterior part of the flank (vs. four dark bands on flank), a smaller eye in males (eye diameter 4—5% SL, vs. 6-7), in having more scale rows between the anal-fin origin and the lateral line (18—22, vs. 15-18). Salmo baliki further differs from S. platycephalus and S. labecula by having fewer gill rakers on the first gill arch (16-18, vs. 21-25), a shorter head in males (head length 24-27% SL, vs. 27—29) and the presence of red spots in specimens larger than about 70 mm SL (vs. ab- sence). In S. baliki, the top of the head is not flattened, while the top of the head is flattened in S. platycephalus, Salmo baliki further differs from S. chilo by the dorsal profile of the head being straight in the interorbital area and at the level of the nostrils (vs. strongly convex), the snout slightly pointed in the male (vs. blunt), the max- illa and lower lip are not fleshy (vs. flesh), fewer black spots behind the eye (always one, vs.up to 12) and fewer black spots on the opercle (3-7, vs. 7-13). Salmo baliki also differs from S. chilo by the number and position of the black spots on the body in males. In S. baliki, black spots are fewer (less than 30) and located on the upper part of the flank. In S. chilo, there are numerous (more than 40) black spots which are scattered on the middle part of the body, mostly on the anterior part; however, these are missing on the back in specimens larger than 140 mm SL. Salmo baliki further differs from S. opimus by having a slenderer body in male (23—26% SL, vs. 26—29), a shorter maxilla in male (maxilla length 8—9% SL, vs. 9-10) with the black circular (vs. irregularly shaped) spots. Addition- ally, the top of the head 1s straight in male (vs. convex) and the mouth is located terminally or slightly subtermi- nal in male (vs. conspicuously subterminal). Salmo baliki is most notably distinguished from S. caspi- us (from Kura River drainage) by having fewer gill rakers on the outer side of the first gill arch (16-18, vs. 19-21) and no black spots on the top of the head (vs. small black spot on top of head). It further differs from S. caspius by having a greater distance between adipose and caudal fins in male (15-18% SL, vs. 14-15), a shorter head in male (24-27% SL, vs. 27-31), a shorter and narrower maxilla in male (maxilla length 8-9% SL, vs. 9-11; maxilla width 2-3% SL, vs. 3-4). In the male specimen of Salmo baliki, the anal- and adipose-fins do not reach the caudal-fin base (vs. reaching in specimens larger than 200 mm SL) and the general body color is silvery in live (vs. brownish). Zoosyst. Evol. 97 (2) 2021, 471-482 Salmo baliki differs from S. rizeensis by the general body color being silvery in live (vs. brownish) and the absence of black spots on the back (vs. presence). Salmo baliki also differs from S. rizeensis by having more scale rows between anal-fin origin and lateral line (24—28, vs. 18-22), less branched dorsal-fin rays (7-9, mode 9, vs. 9-12, mode 10), a shorter head 1n male (24—27% SL, vs. 29-31), a deeper caudal peduncle (11-12% SL, vs. 10- 11), a greater adipose fin (length of base of adipose-fin 45% SL, vs. 3-4), a smaller maxilla in male (length of maxilla 8—9% SL, vs. 10-12), and a smaller mouth gape in male (length of mouth 12-14% SL, mean 12.8, vs. 13—18, mean 15.5). Salmo baliki is immediately distinguished from S. coruhensis by the number and distribution of the black and red spots on the body and the way they vary with increasing size. In S. baliki, the black spots are few, small, ocellated and restricted to the upper part of the flank and missing on the back. The red spots are few, large, irreg- ularly shaped and scatter on the half of the lower and the upper, and median part of the flank. The number of black spots does not increase with increasing size. In S. coruhensis, the black spots are numerous, from medium to large, ocellated, and present on the whole upper half of the flank and on the anterior part of the lower half. The red spots are irregularly shaped and ocellated, and do not increase with increasing size. The number of both kinds of spots increase with increasing size and age. Sal- mo baliki usually has a single pale black spot behind the eye (on cheek and preopercle) at all sizes in both sex; 3 to 7 spots on the opercle. Salmo coruhensis has two or three spots on the cheek and the preopercle in most specimens, rarely a single one spot found and this number increases to 4-17 in large adult male; 5—14 spots on the opercle. Salmo baliki is also distinguished from S. trutta by having fewer lateral line scales (107-118, vs. 117-128), fewer scale rows between lateral line and dorsal—fin origin (27-30, vs. 30-34), a shorter head in males (24-27% SL, vs. 28-31), a smaller maxilla (length of maxilla 8-10% SL, vs. 10-12), a smaller mouth gape in males (length of mouth gape 11-14% SL, vs. 14-16), a deeper caudal peduncle (11-12, vs. 10-11) and greater adipose-fin in males (8-9% SL, vs. 7-8). Salmo baliki further differs by body color and pattern. In S. baliki, red spots are large (greater than eye diameter, vs. smaller than eye diameter) and irregular-shaped (vs. roundish); black spots are few (less than 30, vs. more than 70) and scatter on upper part of flank (vs. numerous and scatter on back, upper part and middle part of flank, sometimes lower part of flank). Results of genetic work was in correspondence with the morphological observations. Although a single mtDNA re- gion (Cyt 6b) was used to assess phylogenetic relationship among Sa/mo sp. in comparison with S. baliki, support of high bootstrap and posterior probability values indicated separation among Sa/mo species inhabiting Anatolia. Salmo sp. are known to be recently diverged (Lobon-Cervia 2018 and references therein) within the course of evolution, thus diversification of species is still an ongoing process, and 481 mostly supported by lower genetic distances among spe- cies. However, phylogenetic analysis of the present study indicated a separate branch for Salmo baliki, when com- pared with the S. euphrataeus, S. fahrettini, S. munzuricus inhabits the same basin. Additionally, each of these species formed unique haplotypes, supporting differences among closely related species of S. munzuricus and S. okumusi. Comparative material See Turan et al. (2010, 2011, 2012, 2014a, 2017, 2020) for additional comparative materials examined. Salmo munzuricus: FFR 3235, 13, 170-253 mm SL; Tur- key: Agri province: stream Alakoglu, a tributary of Eu- phrates River at Taslicay, 39.475000°N, 43.267000°E. —FFR 3241, 4, 108-205 mm SL; Turkey: Mus prov- ince: stream Mengel at Alabalik village, a tributary of Murat River, 39.313686°N, 41.162689°E. —FFR 3226, 11, 123-211 mm SL; Turkey: Tunceli province: stream Kalan at Saritas village, a tributary of Munzur River, 39.249975°N, 39.489062°E. Salmo trutta: Germany, 7, 111-156 mm SL; Rhine River, Plesibach Stream at Niederpleiss. Acknowledgements Many thanks to Taslicay Mayor Ismet Tasdemir (A3r1) for his help arranging the fieldwork in Sinek highland by providing a terrain vehicle. We also would like to thank Baran Yogurtcuoglu (Ankara), Mahmut IItir (Mus), Cev- det Kaya (Bitlis), and Tasligay Municipality employees Metin Tasdemir, Necati Tasdemir and Giray Turan (A&r1) for their help during the fieldwork. Finally, we would like to thank Dr. Greta Carmona Antofianzas (Scotland) for great language editing work on this manuscript. References Bardakc1 F, Degerli N, Ozdemir O, Basibuyuk BB (2006) Phylogeogra- phy of the Turkish brown trout Sa/mo trutta L.: mitochondrial DNA PCR-RFLP variation. Journal of Fish Biology 68(Supplement A): 36-55. https://doi.org/10.1111/j.0022-1112.2006.00948 x Bernatchez L (2001) The evolutionary history of brown trout (Salmo trutta L.) inferred from phylogeographic, nested clade, and mis- match analyses of mitochondrial DNA variation. Evolution 55: 351-379. https://do1.org/10.1111/j.0014-3820.2001 .tb01300.x Berrebi P, Tougard C, Dubois S, Shao Z, Koutseri I, Petkovski I, Crivelli AJ (2013) Genetic Diversity and Conservation of the Prespa Trout in the Balkans. International Journal of Molecular Sciences 14: 23454— 23470. https://doi.org/10.3390/1jms 141223454 Créte-Lafreniere A, Weir LK, Bernatchez L (2012) Framing the Sal- monidae family phylogenetic portrait: a more complete picture from increased taxon sampling. PLoS ONE 7(10): 1-19. https://doi. org/10.1371/journal.pone.0046662 zse.pensoft.net 482 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. Kaya C (2020) The first record and origin of Salmo trutta populations es- tablished in the Upper Tigris River and Lake Van Basin, Eastern Ana- tolia (Teleostei: Salmonidae). Journal of Anatolian Environmental and Animal Sciences 5(3): 366-372. https://do1.org/10.35229/jaes.777575 Kosswig C (1955) Zoogeography of the near East. Systematic Biology, 4: 49-73. https://doi.org/10.2307/sysbio/4.2.49 Kumar S, Stecher G, Li M, Knyazi C, Tamura K (2018) MEGA X: mo- lecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35: 1547-1549. https://doi. org/10.1093/molbev/msy096 Lobon-Cervia J (2018) Princess of the streams: the brown trout Salmo trutta L. as aquatic royalty. In Lobén-Cervia J, Sanz N (Eds) Brown trout — Biology, ecology and management. Hoboken, NJ: Wiley, 1-13. https://doi.org/10.1002/9781119268352.ch1 Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 Snoj A, Mari¢ S, Bajec SS, Berrebi P, Janjani S, Schoffmann J (2011) Phylogeographic structure and demographic patterns of brown trout in North-West Africa. Molecular Phylogenetics and Evolution 61: 203-211. https://doi.org/10.1016/j.ympev.2011.05.011 Suarez J, Bautista JM, Almodovar A, Machordom A (2001) Evolution of the mitochondrial control region in Palaearctic brown trout (Salmo trut- ta) populations: the biogeographical role of the Iberian Peninsula. He- redity 87: 198-206. https://doi.org/10.1046/j.1365-2540.2001.00905.x SuSnik S, Schoffmann J, Weiss S (2005) Genetic verification of native brown trout from the Persian Gulf (Catak Cay River, Tigris basin). Journal of Fish Biology 67: 879-884. https://doi.org/10.1111/j.0022- 1112.2005.00780.x SuSnik S, Knizhin I, Snoj A, Weiss S (2006) Genetic and morpholog- ical characterization of a Lake Ohrid endemic, Salmo (Acantho- lingua) ohridanus with a comparison to sympatric Salmo trutta. Journal of Fish Biology 68: 2—23. https://doi.org/10.1111/j.0022- 1112.2006.00902.x Sekercio3lu CH, Anderson S, Akcay E, Bilgin R, Can OE, Semiz G, Tavsano$lu C, Yokes MB, Soyumert A, Ipekdal K, Sa’ IK, Yiicel M, Dalfes HN (2011) Turkey’s globally important biodiversity in crisis. Biological Conservation 144: 2752-2769. https://doi.org/10.1016/). biocon.2011.06.025 Tamura K, Nei M (1993) Estimation of the number of nucleotide sub- stitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10(3): 512-526. https://doi.org/10.1093/oxfordjournals.molbev.a040023 zse.pensoft.net Turan, D. et al.: Contribution to the Euphrates trout Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position—specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. https://dol. org/10.1093/nar/22.22.4673 Tougard C, Justy F, Guinand B, Douzery EJP, Berrebi P (2018) Salmo macrostigma (Teleostei, Salmonidae): nothing more than a brown trout (S. “rutta) lineage? Journal of Fish Biology 93: 302-310. https://doi.org/10.1111/jfb.13751 Turan D, Kottelat M, Engin S (2010) Two new species of trouts, res- ident and migratory, sympatric in streams of northern Anatolia (Salmoniformes: Salmonidae). Ichthyological Exploration of Fresh- waters 20(4): 333-364. https://www.pfeil-verlag.de/wp-content/up- loads/2015/05/ief20_4 06.pdf Turan D, Kottelat M, Bektas Y (2011) Salmo tigridis, a new species of trout from Tigris River, Turkey (Teleostei: Salmonidae). Zootaxa 2993: 23-33. https://doi.org/10.11646/zootaxa.2993.1.2 Turan D, Kottelat M, Engin S (2012) The trouts of the Mediterranean drainages of southern Anatolia, Turkey, with description of three new species (Teleostei: Salmonidae). Ichthyological Exploration of Freshwaters 23(3): 219-236. https://www.pfeil-verlag.de/wp-con- tent/uploads/2015/05/1ef23_3_03.pdf Turan D, Kottelat M, Engin S (2014a) Two new species of trouts from the Euphrates drainage, Turkey (Teleostei: Salmonidae). Ichthyo- logical Exploration of Freshwaters 24(3): 275-287. http://scholar- bank.nus.edu.sg/handle/10635/128707 Turan D, Dogan E, Kaya C, Kanyilmaz M (2014b) Salmo kottelati, a new species of trout from Alakir Stream, draining to the Mediterra- nean in southern Anatolia, Turkey (Teleostei, Salmonidae). Zookeys 462: 135-151. https://doi.org/10.3897/zookeys.462.8177 Turan D, Kottelat M, Kaya C (2017) Salmo munzuricus, a new spe- cies of trout from the Euphrates River drainage, Turkey (Teleostet: Salmonidae). Ichthyological Exploration of Freshwaters 28: 55-63. https://pfeil-verlag.de/wp-content/uploads/2017/07/ief28_1_04.pdf Turan D, Kalayci G, Bektas Y, Kaya C, Baycelebi E (2020) A new spe- cies of trout from the northern drainages of Euphrates River, Tur- key (Salmoniformes: Salmonidae). Journal of Fish Biology 96(6): 1291-1545. https://doi.org/10.1111/jfb.14321 Turan D, Baycelebi E (2020) First Record of Salmo pelagonicus Kara- man, 1938 (Teleostei: Salmonidae) in the Karamenderes River, Tur- key. Journal of Anatolian Environmental and Animal Sciences 5(4): 551-555. https://doi.org/10.35229/jaes.777776 Warheit KI, Bowman C (2008) Genetic structure of kokanee (Oncorhyn- chus nerka) spawning in tributaries of Lake Sammamish, Washing- ton. Report to King County Department of Natural Resources and Parks, Water and Land Resources Division, and Trout Unlimited — Bellevue/Issaquah, 50 pp. http://your.kingcounty.gov/dnrp/library/ water-and-land/salmon/kokanee/warheit-genetics-report-062308. pdf