Zoosyst. Evol. 100 (1) 2024, 87-99 | DOI 10.3897/zse.100.112557 yee BERLIN The trouts of the Marmara and Aegean Sea drainages in Ttirkiye, with the description of a new species (Teleostei1, Salmonidae) Davut Turan‘, Esra Baycelebi', Sadi Aksu*, Miinevver Oral? 1 Recep Tayyip Erdogan University, Faculty of Fisheries and Aquatic Sciences, 53100 Rize, Turkiye 2 Eskisehir Osmangazi University, Vocational School of Health Services, 26040, Eskisehir, Turkiye https://zoobank. org/6A F795 D4-BBB3-402A-AAAB-IDDS5AE61E435 Corresponding author: Mtinevver Oral (munewver.oral@erdogan.edu.tr) Academic editor: Nicolas Hubert # Received 11 September 2023 Accepted 8 December 2023 @ Published 26 January 2024 Abstract The taxonomic status of native trout species of the Marmara and Aegean Sea drainages is evaluated and three species, Salmo duhani, S. coruhensis and S. brunoi sp. nov., are recognized. Salmo brunoi, a new species, is described from the Niltifer River, a tributary of the Susurluk River. It is distinguished by a general brownish body color in life; few black spots (fewer than 60) on the body, generally scattered on the back and the upper part of the flank, rarely in the median part; few (fewer than 40) and small (smaller than pupil) red spots on the body, scattered on the median part and lower half of the flank; a number of black and red spots not increas- ing with size in both sexes; a long adipose fin (adipose-fin height 8—9% SL); a short distance between adipose-fin and caudal-fin (12-14% SL); and a short anal fin (anal-fin height 12-15% SL). Salmo brunoi sp. nov. is separated from the rest of the Marmara and Aegean trouts of Anatolia based on genome-wide distributed 187.385 unlinked SNP markers. According to the best of the authors’ knowledge, whole genome data is used for the first time here to characterize a new species of trout. Key Words Anatolia, biodiversity, freshwater fish, Salmo, taxonomy Introduction Salmo trutta Linnaeus, 1758 (brown trout) has long been considered a species widely distributed throughout Eu- rope, reaching the Atlas Range southwards (Morocco, Al- geria) and the upper Amu Darya drainage in Afghanistan eastwards (Kottelat and Freyhof 2007). Anatolia has a high level of species richness and en- demism and thus has been classified as a European bio- diversity hotspot (Kosswig 1955; Durand et al. 2002; Sekercioglu et al. 2011), and salmonids are no exception with a high level of endemism in the area (Bardakc¢1 et al. 2006). As it is conveniently located at the intersection of three major biodiversity hotspots, namely: Caucasian, Mediterranean and Irano-Anatolia, Turkiye harbors a high genetic and morphological diversity across a wide range of taxa (Noroozi et al. 2019). So far, the rich di- versity of Anatolian trouts has been mostly revealed by the examination of morphological characters (Tortonese 1955; Behnke 1968; Turan et al. 2010, 2011, 2012, 2014a, 2014b, 2017, 2021, 2022; Turan and Baycelebi 2020; Turan and Aksu 2021) and more recently based on the joint use of genetic and morphological characters (Turan et. al. 2010, 2020, 2021; Kaya, 2020). With these compre- hensive studies, there are seventeen Salmo species natu- rally distributed in Turkiye. These are: Salmo abanticus Tortonese, 1954 (Lake Abant), Salmo araxensis Turan, Kottelat & Kaya, 2022 (Aras River), Salmo ardahanensis Turan, Kottelat & Kaya, 2022 (upper drainages of Kura River), Salmo baliki Turan, Aksu, Oral, Kaya & Bayc¢ele- bi, 2021 (upper drainages of Murat River, Euphrates drainage), Salmo chilo Turan, Kottelat & Engin, 2012 (Ceyhan River), Salmo coruhensis Turan, Kottelat & En- gin, 2010 (the streams and rivers from the Turkish Black Sea coast and Marmara drainages), Salmo duhani Turan & Aksu, 2021 (Gonen Stream, south western Marmara 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. 88 drainage) Salmo euphrataeus Turan, Kottelat & Engin, 2014 (Karasu River, northern Euphrates drainage), Sa/- mo fahrettini Turan, Kalayci, Bektas, Kaya & Bayc¢elebi, 2020 (Karasu River, northern Euphrates drainage), Salmo kottelati Turan, Doan, Kaya & Kanyilmaz, 2014 (Alakir Stream, Mediterranean drainage), Salmo labecula Tur- an, Kottelat & Engin, 2012 (lower drainages of Seyhan River), Salmo munzuricus Turan, Kottelat & Kaya, 2017 (Munzur Stream, northern Euphrates drainage), Salmo murathani Turan, Kottelat & Kaya, 2022 (Aras River), Salmo okumusi Turan, Kottelat & Engin, 2014 (Tohma and Goksu streams, western Euphrates drainage), Salmo opimus Turan, Kottelat & Engin, 2012 (Alara Stream, Mediterranean drainage), Salmo platycephalus Behnke, 1968 (upper drainages of Seyhan River), Salmo rizeen- sis Turan, Kottelat & Engin, 2010 (Turkish Black Sea coast) and Salmo tigridis Turan, Kottelat & Bektas, 2011 (Tigris River) (Turan et al. 2022). Of these, nine species (S. abanticus, S. araxensis, S. ardahanensis, S. coruhen- sis, S. duhani, S. euphrataeus, S. fahrettini, S. murathani, S. rizeensis) belong to the Danubian lineage, while S. tigridis belongs to the Tigris lineage and the remaining species belong to the Adriatic lineage. A previous phylogenetic study of the brown trout based on mitochondrial DNA sequences revealed five major brown trout evolutionary lineages including AD (Adriatic origin), AT (Atlantic), DA (Danubian), MA (Marmaratus), and ME (Mediterranean) (Bernatchez 2001). Subsequently, new lineages were described from Spain as Duero (DU; Suarez et al. 2001), from Turkiye as TI (Tigris; SuSnik et al. 2005; Bardakc¢i et al. 2006), from Morocco as Dades (Snoj et al. 2011), from Northern A fri- ca (Tougard et al. 2018). A molecular study of the brown trout populations inhabiting the Marmara Sea drainages (S. coruhensis and S. duhani) placed them in the DA lin- eage (Bardakci et al. 2006). Next-generation sequencing (NGS) technologies have revolutionized genomic research, enabling the identifi- cation of a massive number of genome-wide markers in a single reaction (Metzker 2010; Goodwin et al. 2016; McCombie et al. 2019). Advances have dramatically reduced the cost while providing high-quality sequence data. NGS has been extensively used in aquatic species, including population structure analysis (Segherloo et al. 2021), genetic linkage map construction (Leitwein et al. 2017), quantitative trait locus mapping for economically important traits (Palaiokostas et al. 2013, 2015) and im- proving the quality of reference genome assemblies (Lien et al. 2016; Hansen et al. 2021). Based on current knowledge, only two valid species, namely Salmo coruhensis and S. duhani, inhabit the riv- ers flowing to the Marmara Sea. Salmo coruhensis 1s distributed in the drainages of the Southern Black Sea and the northern part of the Marmara drainages [Elmal1 Stream (iznik Lake drainage) and Kurtkoy Stream]. Salmo duhani 1s restricted to the upper part of Gonen Stream, the southern drainage of the Marmara Sea. During the present study, additional populations of Salmo were discovered in the Niltifer River (another drainage of zse.pensoft.net Turan, D. et al.: Contribution to the Nilufer trouts the Marmara Sea) and cannot be reliably assigned to one of the two known species from the area. To determine their taxonomic status, we compared their morphologi- cal characters and genome-wide molecular data to other known Salmo species in the area. In addition, the status of the Salmo populations from the Ayazma Stream is reex- amined here. Our comparisons indicate that Salmo popu- lations from the Niltifer River correspond to a distinct and undescribed species belonging to the DA lineage. Materials and methods The fieldwork followed the guidelines of the Local Ethics Committee of RTE University related to the use of an- imals in scientific experiments with a permit reference number of 2014/72. Samples were collected from the streams Aras, Ericek and Delicay, drainages of the Mar- mara Sea and western Turkiye (Fig. 1). These are known to be the uppermost tributaries of the Nilifer River. Speci- mens were captured using an electrofishing device (Samus 1000) and euthanized using tricaine methanesulphonate solution (MS-222). Subsequently, fin clips were collected from one of the pelvic fins and placed into 96% ethanol for subsequent molecular work. Finally, specimens were fixed in a 4% formaldehyde solution in a vertical position. These specimens were deposited at the FFR, Zoology Museum of the Faculty of Fisheries, Recep Tayyip Erdo- gan University, Rize (Sabaj 2020) FSJE, Fischsammlung J. Freyhof, Berlin for detailed morphologic analysis. Abbreviations: SL: Standard length; HL: Head length. Morphological analyses The study by Turan et al. (2010) was used as a guide- line for morphometric analysis. All measurements were carried out in the form of a point-to-point approach (pro- jections were not used) using a dial calliper calibrated to 0.01 mm. Specific to the present study, the last two branched rays articulating on a single pterygiophore in the anal and dorsal fins were counted as “1/2”. Comparison material All materials are from Turkiye except Salmo labrax. Salmo abanticus: FFR 3163, 7,77—-272 mm SL; Bolu prov.: outlet of Abant Lake, 40.5737°N, 31.2957°E. Salmo ardahanensis: FFR 3164, 10, 154-217 mm SL; Ardahan prov.: stream Toros, Kura River drainage, 41.1000°N, 42.4333°E—FFR 3107, 4, 156-192; FFR 3167, 2, 155—182 mm SL; Ardahan prov.: stream Ala- balik, Kura River drainage, 41.0500°N, 42.3666°E.— FFR 3110, 4, 67-118 mm SL; Ardahan prov.: stream Karaman at Asikztlal, Kura River drainage, 41.4166°N, 42.6500°E.—FFR 3136, 16, 99-185 mm SL; Ardahan prov.: stream Kinavur at Cataldere, Kura River drainage, 41.1833°N, 42.6000°E. 41°0'0"N 40°0'0"N 26°0'0"E Zoosyst. Evol. 100 (1) 2024, 87-99 26°0'0"E 27°0'0"E 28°0'0"E Figure 1. Distributions of Sa/mo in Marmara and Aegean Sea basins. Salmo araxensis: FFR 3114, 12, 116-201 mm SL; Kars prov.: Susuz district Kayalik stream, a tributary of Kars stream, Aras River drainage, 40.8166°N, 43.1166°E.—FFR 3115, 15, 93-237 mm SL; Kars prov.: Susuz district: Porsuklu (Akgal1) stream, a tributary of Kars stream, Aras River drainage, 40.8000°N, 43.1833°E.—FFR 3118, 6, 95-132 mm SL; Kars prov.: Sarikamis district: Boyali stream, a tributary of Kars stream, Aras River drainage, 40.4333°N, 42.5666°E.—FFR 3144, 16, 87-265 mm SL; Kars prov.: Susuz district: Incilipinar stream, a tributary of Kars stream, Aras River drainage, 40.8166°N, 43.0666°E. Salmo baliki: FFR 3234, 6, 132-276 mm SL; Agri prov.: stream Sinek a tributary of Murat River at Taslicay, 39.7587°N, 43.4644°E—FFR 3205, 3, 175-267 mm SL; Aéri prov.: a tributary of Murat River 39.7307°N,43.4818°E. Salmo chilo: FFR 3055, 23, 65-235 mm SL; Sivas prov.: stream Akdere at Giurtin, Ceyhan River drainage, 38.6088°N, 36.8962°E. Salmo coruhensis: FFR 3004, 16, 95-240 mm SL; Artvin prov.: stream Osmaniye at Karaosmani- ye village, 41.4689°N, 41.5105°E—FFR 3011, 11, 90-189 mm SL; Artvin prov.: stream Hopa at Cavuslu village, 41.4509°N, 41.7001°E—FFR 3021, 25, 90— 520 mm SL; Rize prov.: stream Firtina at Cat village 40.8653°N, 40.9311°E—FFR 3022, 9,95—228 mm SL; Rize prov.: stream Kendirli at Kalkandere District on road to Kendirli village, lyidere drainage 40.9373°N, 40.4320°E.—FFR 3023, 13, 120-450 mm SL; Rize prov.: stream Iyidere (Ikizdere) at Giineyce 40.8219°N, 40.4765°E.—FFR 3024, 13, 115-330 mm SL; Artvin prov.: stream Dortkilise at Tekkale village, Coruh River, 40.7877°N, 41.4946°E—FFR 3025, 13, 80-550 mm SL; Erzurum prov.: stream Cayirbasi (Kirik) at Kuirik village, Coruh River, 40.2904°N, North Marmara Basin 4 Z Pee, ie we -, ae O"E 28°0'0"E 89 29°0'0"E 30°0'0"E 41°0'0"N *C_) Basin 4 Elevation Value High : 2531 40°0'0"N -Low : -31 © S. brunoi be. A S. duhani se eae os Zag ® S. coruhensis, 29°0'0"E 30°0'0"E 40.8097°E.—FFR 3026, 6, 160-290 mm SL; Er- zurum prov.: stream Buytk at Butytkkoy village, Coruh River, 40.4452°N, 40.8513°E—FFR 3027, 6, 130-420 mm SL; Rize prov.: stream Velikoy at Ve- likoy village, 41.0332°N, 40.6145°E—FFR 3029, 6, 130-220 mm SL; Rize prov.: stream Bozukkale at Bo- zukkale village, 41.0543°N, 40.6297°E—FFR 3030, 6, 80-170 mm SL; Rize prov.: stream Caglayan at Caglayan district 40.9230°N, 40.4452°E—FFR 3031, 6, 190-265 mm SL; Bayburt prov.: stream Olcer at Olcer village, Coruh River, 40.5147°N, 40.5609°E — FFR 3032, 16, 70-310 mm SL; Rize prov.: stream Sogutlt at Sogutlt village, about 5 km west of Cayell, 41.0659°N, 40.6526°E.—FFR 3033, 16, 110-210 mm SL; Bayburt prov.: stream Kurtbogazi at Kurtbogazi village, Coruh River, 40.1883°N, 40.5033°E—FFR 3034, 16, 70-210 mm SL; Giimiishane prov.: stream Harsit at YaSmurdere, 40.5746°N, 39.8645°E.—FFR 3035, 9, 160-450 mm SL; Sivas prov.: stream Ge- min at Camili, Yesirmak River drainage, 40.0619E 38.0536N.—FFR 3037, 10, 90-380 mm SL; Erzurum prov.: stream Pehlivanl at Pehlivanli village, tributary of Tortum, Coruh River, 40.5176°N, 41.4780°E.—FFR 3041, 10, 115—250 mm SL; Trabzon prov.: stream So- lakli at Taskiran village 40.6722°N, 40.2568°E.—FFR 3042, 6, 95-117 mm SL; Rize prov.: stream Sarayk6oy at Saraykoy village, 41.0190°N, 40.3807°E.—FFR 3043, 5, 130-229 mm SL; Artvin prov.: stream Barhal at Sarigol village, Coruh River, 40.9744°N, 41.4184°E — FFR 3043, 9, 110-223 mm SL; Rize prov.: stream Derepazar1 at Derepazar1 41.0237°N, 40.4293°E. — FFR 3044, 6, 100-250 mm SL; Rize prov.: stream Iy- idere at Iyidere 40.9676°N, 40.3778°E.—FFR 3045, 7, 150-450 mm SL; Rize prov.: stream Firtina at Camlihemsin 41.0517°N, 41.0032°E—FFR3046, 5, 10-280 mm SL; Rize prov.: stream Limank6y at L1- mankoy village, 41.0714°N, 40.7121°E. zse.pensoft.net 90 Salmo duhani: FFR 3184, 15, 95-287 mm SL; Canakkale prov.: stream Zeytinli about 9 km east of KazdaS1 Nation- al Park, 39.750°N, 27.017°E, 28.11.2006. -FFR 3185, 14, 85-170 mm SL; Canakkale prov.: stream Zeytinl1, 39.749°N, 27.015°E—FFR 3186, 12, 108-160 mm SL; Canakkale prov.: stream Zeytinli 39.759°N, 27.021°E. FFR 3194, 10, 62-122 mm SL; Canakkale prov.: stream Kocagay1, 12 km west of Kalkim, 39.804°N, 27.071°E.— FFR 3195, 15, 93-275 mm SL; Canakkale prov.: stream Kocagay1 at Yenice, 39.817°N, 27.099°E. Salmo euphrataeus: FFR 1220, 24, 80-260 mm SL; Erzurum prov.: stream Kuzgun, a tributary of Kara- su Stream, Euphrates River drainage, 40.2198°N, 41.1051°E—FFR 1255, 25, 88—230 mm SL; Erzurum prov.: stream Senyurt at Senyurt, a tributary of Karasu Stream, Euphrates River, 40.1830°N, 41.5037°E— FFR 1223, 5, 122—222 mm SL; Erzurum prov.: stream Sirli, a tributary of Karasu Stream, Euphrates River, 40.2183°N, 41.1010°E—FFR 1269, 8, 117-198 mm SL; Erzurum prov.: stream Kuzgun, Euphrates River, 40.2198°N, 41.1050°E. Salmo fahrettini: FFR 3232, 20, 134-227 mm SL; Er- zurum prov.: stream Omertepesuyu at Palandoken 39.7958°N, 40.9444°E. —FFR 3233, 5, 126-194 mm SL; Erzurum prov.: stream Tekke at Palandoken, 39. 8197°N, 41.1516°E. Salmo kottelati: FFR 3181, 21, 98-210 mm SL; An- talya prov.: stream Alakir at Altinyaka, 36.5608°N, 30.3428°E—FFR 3182, 16, 98-176 mm SL; Antalya prov.: stream Alakir at Alttnyaka, 36.5608°N, 30.3428°E. Salmo labecula: FFR 3057, 4, 103—237 mm SL; Nigde prov.: stream Ecemis at Camardi, Seyhan Riv- er drainage, 37.8253°N, 34.9902°E—FFR 3058, 5, 142-241 mm SL; Isparta prov.: stream Kartoz at AsaSiyaylabel, Kopriicay drainage, 37.5532°N, 31.3070°E—FFR 3059, 5, 140-184 mm SL;Antalya prov.: stream Zindan at Aksu, Koprticay drainage, 37.8064°N, 31.0734°E. Salmo labrax: FSJF 396, 6, 107-147 mm SL; Ukraine: Ula—Uzev River; N. Bogustkaya, A. Neseka, J. Bohlen & J. Freyhof, 12 June 2002.—FSJF 10, 6, 102-160 mm SL; Russia: Crasnodar prov.: Khosta River; J. Freyhof, 19July 2002. Salmo munzuricus: FFR 3162, 17, 127-270 mm SL; Tunceliprov.: stream MunzuratKoyungolt,,39.3472°N, 39.1341°E—FFR 3147, 8, 146-320 mm SL; stream Munzur at Koyungoli, 39.3461°N, 39.1316°E. Salmo murathani: FFR 3121, 18, 60-233 mm SL; Kars prov.: Keklik stream [a tributary of Kars stream], Sarikamis district, Aras River drainage, 40.2833°N, 42.6500°E.—FFR 3117, 22, 95-192 mm SL; FFR 3113, 17, 91-206; Kars prov.: Keklik stream [a trib- utary of Kars stream] Sarikamis district, Aras River drainage, 40.2500°N, 42.6666°E—FFR 3120, 10, 69-163 mm SL, Kars prov.: Maksutcuk stream [a trib- utary of Kars stream], Aras River drainage, 40.5333°N, 42.8666°E.—FFR 3108, 14, 90-186 mm SL; Ardahan prov.: Cildir Lake, Aras River drainage 41.0500°N, zse.pensoft.net Turan, D. et al.: Contribution to the Nilufer trouts 43.3166°E—FFR 3228, 23, 95—241 mm SL; Kars prov.: Arpacay stream [a tributary of Kars stream] Arpacay district, Aras River drainage 40.9000°N, 43.1666°E.— FFR 3229, 8, 110-156 mm SL; Kars prov.: Keklik stream [a tributary of Kars stream] Sarikamis District, Aras River drainage, 40.2833°N, 42.6500°E. Salmo okumusi: FFR 1254, 10, 75-202 mm SL; Malatya prov.: stream Strgu, Euphrates River drainage, 37. 997T5°N, 37.9583°E—FFR 125, 10, 129-169 mm SL; Sivas prov.: stream Gokpinar, a tributary of Tohma stream, Euphrates River, 38.6600°N, 37.3089°E.— FFR 1256, 10, 68-280 mm SL; Sivas prov.: stream Gokpinar, Euphrates River, 38.6600°N, 37.3089°E.— FFR 124, 2, 149-175 mm SL; Kahramanmaras prov.: stream Goksu 4 km north of Dizbag, Euphrates River, 37.8331°N, 37.4756°E. Salmo opimus: FFR 3048, 12, 118-180 mm SL; Anta- lya prov.: stream Alara at Gundogmus, 36.7921°N, 31.9749°E—FFR 3049, 20, 115-186; Kahraman- maras prov.: stream Goctksu at Komutrkoy, Ceyhan River drainage, 38.1447°N, 36.5630°E.—FFR 3050, 4, 175-210 mm SL; Kahramanmaras prov.: drain- age of stream Tekir at Tekir, Ceyhan River drainage, 37.8767°N, 36.6058°E.—FFR 3051, 9, 90-300 mm SL; Kahramanmaras prov.: stream Firniz at Firniz, Ceyhan River drainage, 37.7591°N, 36.6983°E. Salmo platycephalus: FFR 972, 7, 145-184 mm SL; Kayseri prov.: Pinarbasi stream at Pinarbas1 district, Seyhan River drainage —FFR 1260, 10, 137-237 mm SL; Kayseri prov.: Pinarbasi Stream at Pinarbasi dis- trict, Seyhan River drainage. Salmo rizeensis: FFR 3001, 15, 90-220 mm SL; Er- zurum prov.: stream Ovit (2) [Kan] at Ovit mountain, Coruh River, 40.5887°N, 40.8583°E—FFR 3002, 10, 114-245 mm SL; Trabzon prov.: stream Degirmen at Cosandere village, 40.7512°N, 39.5908°E—FFR 3003, 12, 112-230 mm SL; Trabzon prov.: stream Solakli at Demirkap1 village, 40.7586°N, 40.5913°E—FEFR 3005, 13, 111-220 mm SL; Rize prov.: stream Caglayan at Gurctidtizii plateau 41.1905°N, 41.3086°E—FFR 3006, 18, 95-226 mm SL; Rize prov.: stream Sehitlik at Sehitlik village, 41.1407°N, 40.9828°E—FFR 3007, 12, 90-118 mm SL; Rize prov.: stream Cayeli at Kap- tanpasa village, 40.958°N, 40.7794°E—FFR 3008, 18, 91-198 mm SL; Rize prov.: stream Firtina at Tun- ca village, 41.1259°N, 41.1310°E—FFR 3009, 10, 110-240 mm SL; Rize prov.: stream Taslidere at Pas- acur village; 40.8837°N, 40.5796°E—FFR 3010, 9, 110-240 mm SL; Rize prov.: stream Taslidere at Kangel village, 40.9453°N, 40.6642°E—FFR 3011, 7, 100-180 mm SL; Rize prov.: stream Erenler at Erenler village, 41.0914°N, 40.8298°E—FFR 3012, 7, 88-237 mm SL; Artvin prov.: stream Dortkilise at Tekkale Village, Coruh River, 40.7800°N, 41.5098°E—FFR 3013, 12, 75-167 mm SL; Artvin prov.: Ciftekoprii stream at Cankurtaran mountain, Coruh River, 41.3844°N, 41.5691°E—FFR 3014, 7, 112-201 mm SL; Artvin prov.: stream Kapisre at Ktictkkoy village, 41.2753°N, Zoosyst. Evol. 100 (1) 2024, 87-99 41.3755°E—FFR 3015, 9, 113-228 mm SL; Bayburt prov.: stream Kop at Kop Mountain, Coruh River, 40.0654°N, 40.433 1°E—FFR 3016, 9, 113-221 mm SL; Erzurum prov.: stream Yagli at Yagl village, Coruh Riv- er, 40.3643°N, 41.0728°E—FFR 3017, 12, 112-223 mm SL; Erzurum prov.: stream Buytik at Buytikdere plateau, Coruh River drainage, 40.5698°N, 40.7140°E—FFR 3018, 16, 145-224 mm SL; Gtimishane prov.: stream Akbulak at Akbulak village, Yesilirmak River drainage, 40.281462°N, 39.0896°E.—_FFR 3019, 10, 122-221 mm SL; Kutahya prov.: stream Sefakoy at Domani¢, Sakarya River drainage, 39.8426°N, 29.6706°E—FEFR 3020, 10, 111-119 mm SL; Kitahya prov.: Catalalic Stream at Domanic, Sakarya River, 39.8600°N, 29.6291°E—FFR 3036, 10, 130-170 mm SL; Rize prov.: stream Ikizdere at Anzer plateau, 40.5926°N, 40.5148°E—FFR 3038b, 7, 130-170 mm SL; Rize prov.: stream Ciftekavak at Ortapazar village, 40.9959°N, 40.485 1°E—FFR 3039a, 14, 120-200 mm SL; Rize prov.: stream Firtina at Ele- vit Plateau, 40.8471°N, 41.0151°E—FFR 3038a, 1, 250 mm SL; Erzurum prov.: stream Ovit (2) [Kan] at Ovit mountain, Coruh River, 40.5735°N, 40.8634°E — FFR 3039b, 10, 90-238 mm SL; Rize prov.: stream Ovit at Ovit mountain, Iyidere drainage, 40.6361°N, 40.8214°E—FFR 3040, 14, 90-190 mm SL; Erzurum prov.: stream Merekum at Merekum, Coruh River, 40.5527°N, 41.4592°E. Salmo tigridis: FFR 1253, 9, 136-227 mm SL; Van prov.: stream Catak, Tigris River, 38.0077°N, 43.0652°E. Samples In total, 71 samples fixed in formalin were investigat- ed morphologically (see Paratypes section) and tissue samples were collected from two specimens of the new species, S. brunoi, originating from Bursa, Uludag, Aras Stream, Turkiye. In total, 12 samples were examined for genetic analysis including 2 specimens of new species S. brunoi from Bursa, Uludag, Aras Stream, 1 speci- men of Salmo coruhensis, collected from Bursa, Iznik, Sigirhisar village and 3 specimens of Salmo coruhensis from Sultaniye Stream, Kartepe, [zmit, 3 specimens of Salmo pelagonicus collected from Canakkale, Bayramic, Ayazma Stream and 3 specimens of S. duhani taken from the type locality, in Canakkale, Yenice, Kalkim. In ad- dition to Anatolian samples, globally recognized Salmo lineages were included as references in the genetic analy- ses. From these references 3 specimens of Danubian lin- eage samples included in the genetic analysis (1 specimen provided from the Kuban River, Russia, has been treat- ed as S. /abrax based on Turan et al. (2014b) who have previously reported the distribution of the species from the northwest Caucasia in Russia to the Danube River and 2 specimens from the Sevan River, Armenia were treated as S. ischchan (thus Danubian reference). The rest of the reference samples included 2 specimens from S. marmaratus from Svenica and Trebuscica in Slovenia; 3 specimens from Atlantic fish origin of Babeau hatchery 91 in France (unidentified species), 2 specimens of S. obtu- sirostris from Studen¢éica in Bosna and Herzegovina, 3 specimens of Adriatic lineage samples (unidentified spe- cies) from Alfios and Kalamos in Greece and | specimen from Ohrid-Drin-Skadar in Albania, respectively. DNA extraction, ddRADseq library preparation and NGS sequencing Total genomic DNA extraction was carried out on a King- Fisher Flex DNA extraction robot (Thermo Fisher Scien- tific, France) following the manufacturer’s instructions. DNA quality was assessed on 0.8% agarose gels and DNA quantity was estimated using a NanoDrop 2000 (Thermo Fisher Scientific, France). High molecular weight genomic DNA samples were further assessed using Qubit (Thermo Fisher Scientific, France) BR assay for the final quantifi- cation of double-stranded DNA prior to ddRADseq library construction. The library construction was performed fol- lowing the original ddRADseq protocol by Peterson et al. (2012) with slight modifications detailed by Leitwein et al. (2016) and Oral (2023). Genomic DNA was doubled-di- gested using EcoRI and Msp/ enzymes. Fragmented DNA was then individually barcoded using adaptors. Samples were pooled and processed into single tubes following adaptor ligation. Purified and size-selected fragments (c. 300-700 bp) were then enriched for 15 PCR cycles. The amplified library was quantified using NanoDrop spec- trometry and Qubit fluorimetry and the size distribution of the library was further assessed on a Fragment Ana- lyzer (Advanced Analytical Technologies, France). The ddRADseq library was sequenced on an I/lumina Nova- Seq platform with paired-end reads of 150 base pairs. Bioinformatic data analysis The initial quality control of the raw data files was car- ried out using FastQC (Andrews 2010; Babraham Bio- Informatics). Reads of low quality (Phred score < 30), missing restriction sites and/or involving ambiguous barcodes were removed. Retained reads were then pro- cessed using Stacks v2.55 (Catchen et al. 2013) for de- multiplexing based on their barcodes, restriction enzymes and cleaned with ProcessRadtags (-c -r -q --renz_1 eco- RI --renz_2 mspI). Cleaned reads were mapped against the Salmo trutta reference genome (accession number: GCA_901001165.2; Hansen et al. 2021) with BWA- mem2 v2.1 (-k 19 -c 500 -O 0,0 -E 2,2 -T 0 -R) (Li and Durbin 2010) and samtools v1.11 (-Sb -q 1 -F 4 -F 256 -F 2048). Then gstacks (--max-clipped 0.01) was run with a minimum number of 2 populations where a locus must be present (-p 2), a minimum 20% of individuals in a pop- ulation (-r 0.2), a maximum observed heterozygosity of 60% (--max-obs-het 0.6), a minimum allele frequency of 1% (--min-maf 0.01) and a single representative of each overlapping site (--ordered-export). zse.pensoft.net 92 Once variants were collected following the steps above mentioned, they were filtered with vcftools v0.1.16 (Danecek et al. 2011). First, we focused on individuals, removing those with more than 20% of missing data. Sec- ond, we filtered SNPs according to the sequencing depth, missing data, frequency and number of alleles per site (--minDP 4 --minGQ 30 --max-missing 0.4 --min-alleles 2 -max-alleles 2 --maf 0.01). Finally, we removed SNPs that were in high linkage disequilibrium using 11_ex- tract_unlinked_snps_genome.py (diff_threshold=0.5 and max_distance=50) from stacks workflow v2.62 (https:// github.com/enormandeau/stacks workflow). Buoinfor- matics analyses were performed with the support of LD- genX (www.Idgenx.com) and only a subset of these data was used in the present study. Population structure analysis We performed ADMIXTURE and Principal Component Analysis (PCA) on filtered and unlinked SNPs. AD- MIXTURE v.1.3.0 (Alexander et al. 2009) was used to estimate individual cluster memberships. ADMIXTURE provides an estimation of individual ancestry proportion for K groups and the number of different groups was ex- plored from 1 to 12. Based on the cross-validation pro- cedure, the best K with the lowest cross-validation error was detected as 9. Q-values estimated by ADMIXTURE were used to produce bar plots with R v 4.2.1 (R Core Team 2015). Alongside the ADMIXTURE analysis, the unlinked SNPs of 12 individuals from the Marmara Aegean ba- sin were further investigated using PCA calculated with PLINK 1.9 (Chang et al. 2015). PCA was conducted to determine the population structure and the first two com- ponents of the PCA were plotted using R v 4.2.1. Results Salmo brunoi sp. nov. https://zoobank.org/6A B6FDA0-37BF-49D8-8A 74-BE2FCED9212F Figs 2-4 Type material. Holotype: FFR 3243, 175 mm SL; Turki- ye, Bursa prov.: stream Aras, a tributary of Niltifer River, 40.0536°N, 29.1722°E. Paratypes: FFR 3216, 188-153 mm SL; same data as holotype —FFR 3213, 7, 142-195 mm SL;—FFR 3215, 7, 142-195 mm SL; Turkiye, Bursa prov.: stream Delicay at Kestel, 40.1241°N, 29.2737°E—FFR 3211, 18, 93- 180 mm SL; —FFR 3217, 12, 85-153 mm SL; Turkiye, Bursa prov.: stream Ericek at Osmangazi, 40.0426°N, 29.2098°E. Diagnosis. Salmo brunoi is distinguished from all the species of Sa/mo in Turkiye and adjacent areas by the combination of the following characters: a small size (known maximum size 187 mm SL); body brownish zse.pensoft.net Turan, D. et al.: Contribution to the Nilufer trouts in life; one black spot in postorbital and suborbital ar- eas, greater than the pupil; two to four black spots on the opercle, approximately smaller than the pupil; black spots on the body few (fewer than 60), approximately equal to the pupil, ocellated, scattered on the back and the upper part of the flank (missing in the predorsal area); red spots few (fewer than 40), smaller than the pupil, irregularly shaped, surrounded by an irregularly shaped narrow ring, organized in two to four irregular longitudinal rows; number of black and red spots not in- creasing with size; anal fin short (12-15% SL in males, 12-14 in females), adipose fin large (adipose fin height 8—9% SL in males and females), short distance between adipose fin and caudal fin bases (13-14% SL in males, 12-14% in females). Description. The general appearance is shown in Figs 2, 3, live images are in Fig. 4, morphometric data are in Table 1. Body moderately deep, compressed laterally, its depth smaller than head length. The dorsal profile is Slightly arched, and the head is short, upper profile slight- ly convex on the interorbital area and the snout in males and markedly convex on both interorbital areas and the snout in females. Mouth large in males, small in females, terminal or slightly subterminal in males, subterminal in females. Tip of lower jaw slightly curved upwards, point- ed, with a slightly developed process at symphysis in males larger than 160 mm SL. Maxilla somewhat long, with a length of 10-12% SL, reaching beyond the posteri- or margin of the eye in males larger than 140 mm SL and only reaching the posterior margin of the eye in females. Snout somewhat short, with a pointed tip in males, round- ed in females. Adipose fin long, height about 8-9% SL in males and in females. Known maximum size 195 mm SL. Dorsal fin with 3-4 unbranched and 8—10 branched rays, its distal margin convex. Pectoral fin with 1 un- branched and 11-13 branched rays, its external margin slightly convex. Pelvic fin with 1 unbranched and 7-8 branched rays, its external margin convex. Anal fin with 3 unbranched and 7—9 branched rays, its distal margin convex anteriorly and concave posteriorly. The caudal fin deeply emarginated in specimens less than 120 mm SL, slightly emarginated or truncated in specimens larger than 140 mm SL, lobes slightly pointed. Lateral line with 108-122 scales; 23—32 scale rows between dorsal fin or- igin and lateral line; 16—23 scale rows between anal fin origin and lateral line; 14-18 scale rows between origin of the adipose fin and lateral line. Gill rakers 15—18 on the first gill arch. Coloration. In life: General body color brownish or light brownish. Back and flank brownish and belly yel- lowish. Red spots conspicuously organized in two to four irregular longitudinal rows on the median part of the body and half of the lower part of the flank. Conspicuous- ly black spots in postorbital and suborbital areas. Black spots roundish, scattered on back and upper part of flank. Pectoral, pelvic and anal fins yellowish, dorsal and anal fins yellowish or light brownish. Adipose fin with reddish margin (see Fig. 4). Zoosyst. Evol. 100 (1) 2024, 87-99 93 Figure 2. Salmo brunoi, FFR 3243, holotype, 175 mm SL, male; Turkiye: stream Aras, a tributary of Niltifer River. Figure 3. Salmo brunoi, from top: FFR 3216, paratypes, 137 mm SL, male; 105 mm SL, female; Turkiye: stream Aras, a tributary of Niltifer River. In formalin: The general coloration of freshly pre- served specimens dark brown on the back and upper part of the flank, brownish on the lower part of the flank and yellowish on the belly. One black spot in postorbital and suborbital areas, greater than the pupil: two to four black spots on the opercle, approximately smaller than the pu- pil. Black spots on the body few (fewer than 60), approx- imately equal to the pupil, ocellated, commonly scattered on the back and the upper part of the flank (missing in the predorsal area) and rarely median part of the flank; no black spot on top of the head. Red spots few (fewer than AQ), small (smaller than the pupil), irregularly shaped, surrounded by an irregularly shaped narrow ring, orga- nized in two to four irregular longitudinal rows on the median part of the body and half of the lower part of the flank. The number of black and red spots on the flanks do not increase with size. Dorsal fin gray, with two or three rows of black spots (smaller than pupil) and one or two rows of red spots (smaller than pupil). Caudal fin dark gray; pectoral, anal and pelvic fins grayish. Adipose fin plain grayish, rarely one or two red spots on its posteri- or edge (Figs 2,3). Eleven to thirteen parr marks on the body, distinct in specimens up to about 195 mm SL. Distribution and habitat. Sa/mo brunoi sp. nov. in- habits clear and swift-flowing water, with a substrate consisting of gravel and pebbles. The observed materi- al for this species has been collected from streams Aras, Deligay and Ericek, drainages of Niltifer River (Fig. 1). Conservation status. According to the First Author’s (DT) observations, Salmo brunoi sp. nov. 1s under the in- fluence of overfishing. Besides fresh consumption, trout oil is a widely preferred natural remedy, particularly for the treatment of rheumatism, muscle, and joint pains among local people (Turan et al. 2006). Therefore, the zse.pensoft.net 94 Turan, D. et al.: Contribution to the Nilufer trouts Table 1. Morphometry of Salmo brunoi (holotype, FFR 3243; paratypes FFR 3215, n=6, and FFR 3216, n=8). The calculations include the holotype. Holotype Paratypes Sex male male SD female SD Number of specimens n=6 n=8 Standard length (mm) 175 112-179 110-153 In percentage of standard length Range (mean) Range (mean) Head length 29.6 26.1-29.6 (27.7) 13 24.8-26.9 (26.0) 0.7 Predorsal length 49.6 47.1-49.6 (48.7) 0.8 44 .2-48.4 (47.1) 1.4 Prepelvic length 55.9 53.8-55.9 (54.8) 0.8 52.7-55.5 (53.8) 1.0 Preanal length 136 73.2-75.0 (74.3) 0.7 73.3-75.7 (74.2) 1.0 Body depth at dorsal-fin origin 24.8 22.1-25.3 (23.8) 0.4 19.9-24.3 (21.6) 1:3 Body depth at anal-fin origin 19.2 16.2-19.3 (17.7) Ll 16.1-17.8 (16.8) 0.6 Depth of caudal peduncle 10.9 9.9-10.9 (10.1) 0.3 8.8-10.0 (9.4) 0.4 Length of caudal peduncle 17.0 15.5-18.0 (16.6) 0.9 15.3-17.8 (17.0) 0.8 Distance between adipose- and caudal-fins 14.0 12.6-14.4 (13.5) 0.6 11.5-13.6 (12.6) 0.7 Body width at anal-fin origin 9.0 7.1-10.4 (8.9) 1.3 7.0-9.9 (9.0) 1.0 Length of dorsal-fin base 9.0 12.2-14.5 (13.3) 0.9 12.7-13.8 (13.1) C5 Height of dorsal fin 19.4 16.3-19.5 (17.6) 1.2 15.2-17.1 (16.2) 0.7 Length of pectoral fin 14.1 15.9-20.1 (18.0) 1.6 16.3-18.5 (17.3) 0.8 Length of adipose-fin base 3.7 2.9-4.1 (3.6) 0.5 2.8-4.8 (3.8) 0.4 Height of adipose fin 8.6 8.0-9.2 (8.4) 0.5 7.8-8.5 (8.1) 0.2 Length of pelvic fin 19.4 12.1-15.3 (13.4) 1.6 11.9-14.4 (13.1) 0.9 Height of anal fin 13.4 12.1-14.7 (13.4) 1.2 11.9-14.4 (13.1) 0.9 Length of anal-fin base 10.7 9.3-12.2 (10.4) 0.9 8.3-11.6 (10.3) led Length of upper caudal-fin lobe 19.9 12.9-17.6 (15.6) 1.6 15.4-17.7 (16.3) 0.9 Length of median caudalfin rays 14.3 10.9-14.7 (13.0) 1.4 10.8-14.0 (12.4) 1.1 Length of lower caudal-fin lobe 14.7 14.0-18.0 (16.2) 1.6 15.2-18.5 (16.6) ba Snout length 8.8 6.7-8.7 (7.7) 0.8 6.6-7.4 (7.0) 0.3 Distance between nasal openings 4.8 4.0-5.6 (4.9) 0.5 4.0-4.8 (4.4) 0.2 Eye diameter 6.1 5.2-7.0 (6.0) 0.6 5.4-6.6 (5.8) 0.4 Interorbital width 8.4 7.3-8.4 (7.8) 0.4 7.0-8.0 (7.5) 0.3 Head depth through eye 13.4 11.2-13.4 (12.5) 0.8 11.5-13.4 (12.8) 0.6 Head depth at nape L75 15.0-17.7 (16.3) 0.4 16.1-17.9 (16.9) 0.7 Length of maxilla 12.0 9.5-12.0 (10.5) 0.9 8.5-9.7 (9.2) 0.5 Maximum height of maxilla 2.5 2.6-3.7 (3.1) 0.3 2.6-3.8 (3.1) 0.4 Width of mouth gape 9.7 7.9-11.0 (9.2) 1.0 8.0-9.3 (8.7) 0.4 Length of mouth gape 16.6 12.2-16.7 (14.0) 1.6 12.0-13.2 (12.6) 0.5 species is in high demand. Given the highly restricted dis- tribution of S. brunoi sp. nov. to a very limited area (only three streams), and considering the above mentioned so- cio-economic interest, this species is likely to be under a serious threat. Thus, there is a need for the species to be conserved under international legislation. Comparison with other Salmo species. Salmo brunoi sp. nov. differs from the other species of trout recorded from Marmara, Aegean and Black Sea basins (S. duhani, S. coruhensis, S.abanticus, S. rizeensis and S. labrax) by having a shorter anal fin in females (12-14% SL, vs. 14— 20), a longer adipose fin in females (adipose fin height 8—-9% SL, vs. 4-8) and males (8-9% SL, vs. 4-8, except S. coruhensis), a shorter distance between adipose fin and caudal fin bases in females (12-14% SL, vs. 14-17, except S. duhani) and males (13-14% SL, vs. 15-17 in S. labrax, 14-16 in S. rizeensis, 14-16 in S. duhani, ex- cept S. abanticus and S. coruhensis). Salmo brunoi fur- ther differs from S. abanticus, S. coruhensis and S. labrax by the brownish body color in life (vs. silvery). Salmo zse.pensoft.net brunoi further differs from S. abanticus and S. coruhensis by fewer black spots on the body in adult males (fewer than 60, vs. more than 80), whose number does not in- crease with size (vs. number increasing with size). Sal- mo brunoi further differs from S. duhani by having fewer black spots on the back and flank in females (fewer than 60, vs. more than 80). Salmo brunoi is further distin- guished from S. abanticus by the presence of red spots on the body in all sizes (vs. absent in specimens larger than about 200 mm SL) and black spots circular (vs. polygo- nal).Salmo brunoi is further distinguished from S. /abrax by having a shorter predorsal distance in males (47-50% SL, vs. 46-47), a slenderer body in males (body depth at anal fin origin 16-19% SL, vs. 19-21) and a slender- er caudal peduncle in females (9-10% SL, vs. 10-11). Salmo brunoi is further distinguished from S. rizeensis by having a slenderer caudal peduncle in females (9-10% SL, vs. 10-11). The new species, Salmo brunoi, is also distinguished from S. ardahanensis by having fewer gill rakers on Zoosyst. Evol. 100 (1) 2024, 87-99 | ame, SA, “<= 5 ~ 95 ™~ Pt Figure 4. Salmo brunoi, from top: not preserved, ~145 mm SL, male; not preserved, ~150 mm SL, female; Turkiye: stream Aras, a tributary of Niltifer River. the outer side of the first gill arch (15-18, vs. 19-21), no black spots on the top of the head (vs. small black spots). It further differs from S. ardahanensis by having a smaller distance between adipose and caudal fins in fe- males (12-14% SL, vs. 14-17) and a shorter anal fin in males (12—15%SL, vs. 15—18). In males of Salmo brunoi, anal and adipose fins do not reach the caudal fin base (vs. reaching in specimens larger than 200 mm SL) and the interorbital area is convex (vs. flat straight). Salmo brunoi is further distinguished from Salmo mu- rathani by having fewer black spots on flank and back in adult specimens (fewer than 60, vs. more than 66); one black spot behind eye (larger than pupil); 2-4 spots on preopercle and opercle (vs. 4-15); black spots scattered on back (missing in predorsal area), the upper part of flank, sometimes a few black spots below lateral line be- hind head (vs. black spots scattered on back, the middle and upper part of the flank and the anterior part of the lower flank in males) black spots few (34-47), restricted to the back and upper part of flank in females smaller than about 210 mm SL). It further differs from S. murathani by having a smaller distance between adipose and caudal fins in females (12—14% SL, vs. 15—17) and a shorter anal fin in females (12-14% SL, vs. 14-18) and a slenderer caudal peduncle depth in females (9-10% SL, vs. 10-12). Salmo brunoi is distinguished from Sa/mo araxensis by having a longer maxilla in males (10-12% SL, vs. 9-10), a Shorter anal fin (12—15% SL in males, 12—14 in females, vs. 15—18 in males, 14-18 in females), a slenderer caudal peduncle in females (9-10% SL, vs.10—12) and a small- er distance between adipose and caudal fins in females (12-14% SL, vs. 14-17). Salmo brunoi is distinguished from S. fahrettini by having the general body color brownish in life (vs. sil- very); fewer black spots on the body (fewer than 60, vs. more than 80); black spots on the back (missing on the predorsal area) and upper part of flank, sometimes a few below lateral line behind the head (vs. black spots scat- tered on back, middle and upper part of flank and anterior part of lower half of flank); their number not increasing with size (vs. their number increasing with size); fewer red spots on body (fewer than 40, vs. more than 70 in adult specimens), their number not increasing with size (vs. increasing with size); a longer maxilla in males (length 10-12% SL, vs. 9-10); a longer adipose fin in males (8—9% SL vs. 3-8); a smaller distance between ad- ipose and caudal fins in females (12-14% SL, vs. 15-18) and a shorter anal fin in females (12-14% SL, vs. 15-17). Salmo brunoi is distinguished from S. euphrataeus by having a smaller distance between adipose and caudal fins in males (13-14% SL, vs. 14-16), a slenderer caudal peduncle in females (9—10% SL, vs.10—12), a shorter anal fin in females (12-14% SL, vs. 16-19), and the adipose fins do not reach the caudal fin base (vs. reaching in spec- imens larger than 200 mm SL). Salmo brunoi is distinguished from S. platycepha- lus, S. chilo, S. labecula, S. kottelati, S. opimus, all from streams draining to the Mediterranean and S. okumusi, zse.pensoft.net 96 S. munzuricus and S. baliki from Euphrates River, by hav- ing a smaller distance between adipose and caudal fins in males (12-14% SL, vs. 14-19), a slenderer caudal pedun- cle in females (9—10% SL, vs.10—13), a shorter anal fin (12-15% SL, vs.15—21, except S. /abecula and S. mun- zuricus) and fewer gill rakers on first gill arch (15-18, vs. 18-25, except S. munzuricus and S. baliki). Salmo brunoi is further differs from S. platycephalus, S. chilo, S. la- becula, S. kottelati and S. opimus, by the absence of four dark bands on the flank (vs. presence). It further differs from S. munzuricus by having a smaller adipose in males (8-9% SL, vs. 9-12) and a longer maxilla in males (10- 12% SL, vs. 8-10). It further differs from S. platyceph- alus by the presence of red spots on flank (vs. absent in specimens larger than about 70 mm SL) also differs from S. labecula by the presence of red spots on flank in all size (vs. absent in specimens larger than about 70 mm SL). Salmo brunoi differs from S. tigridis by having fewer scale rows between the dorsal fin origin and the lateral line (23-32, vs. 32-35); fewer scale rows between the end of the adipose fin base and the lateral line (14-18, vs. 19-20), a slenderer caudal peduncle depth 9-11% SL, vs. 12-13). Sexual dimorphism. The maxillary length in males is longer than that of females (10-12% SL in males, 9-10 in females). The length of mouth gape in males is longer than that of females (12-17% SL, 12-13). The snout of the male is more pointed than that of the female. Etymology. The species is named after Dr. Bruno Guinand (University of Montpellier, ISEM, France) for his valuable contribution to Salmo population genom- ics research. ddRAD loci and SNP calling In total, an average of thirteen million raw reads were generated per individual with a mean sequence depth of 30. Sequences with a missingness index higher than 20% were removed from the dataset. Once filtered according to sequencing depth, missing data, frequency and number of alleles, a total of 215k SNPs were retrieved. More than 187k unlinked SNPs within the 50 bp window were used for downstream population analysis. Interference of ADMIXTURE and PCA analysis The ADMIXTURE program identified 9 separate clus- ters. In the reference lineages, the Danubian (DA) cluster was placed in two groups of which DA-1 (S. /abrax) sep- arated from DA-2 and DA-3 (S. ischchan) corresponding to the origin and the geographic basin. The rest of the reference Salmo species including S. obtusirostris and S. marmaratus clustered separately, as expected. Similar- ly, S. brunoi sp. nov., generated a separate cluster from the rest of the Marmara and Aegean trout of Anatolia. The zse.pensoft.net Turan, D. et al.: Contribution to the Nilufer trouts only exception was observed in Salmo duhani, which 1n- dividuals clustered together (K=9; Fig. 5; see Discussion for detailed explanation). The 187,385 unrelated SNPs for each of the 12 indi- viduals from the Marmara Aegean basin were used for PCA. The analysis results indicated 3 clusters of which the first cluster included DA reference samples originated from Armenia as S. ischchan, the second cluster included S. brunoi and S. coruhensis clustered with S./abrax from Russia and the third cluster included S. duhani and that of S.pelagonicus (see discussion). The first two components of PCA represented 29.31% and 21.39% of the variance among individuals. Discussion Up until the present study, three species of trout have been reported from the Marmara and Aegean Sea drain- ages: S. duhani (Gonen Stream-Marmara Sea drainage), S. coruhensis (Elmali and Kurtkoy streams, Iznik and Sapanca Lake drainages) and S. pelagonicus (Ayazma Stream; Karamenderes drainage, Aegean Sea drain- age). In the present study, our molecular data (Q val- ues, 0.99992, 0.9992 respectively for S. pelagonicus and S. duhani, Fig. 5) showed that trout samples from Gonen Stream (Marmara Sea drainage) largely overlapped in genetic diversity of 187,385 genome-wide SNP mark- ers with those of Ayazma Stream samples (Aegean Sea drainage). Turan and Baycelebi, (2020) reported Ayazma samples as Salmo pelagonicus. Indeed, Salmo pelagonicus was originally described from Mountain Brooks in Macedonia (Karaman, 1938). Although Tur- an and Baycelebi, (2020) compared specimens from the Ayazma stream with 3 photographs of S. pelagoni- cus from the Aliakmon River in Greece, these authors did not compare the Ayazma population with that of the type locality for S. pelagonicus from Macedonia. Later, Turan and Aksu (2021) described Salmo duhani from Gonen Stream and gave a few morphological differenc- es between S. duhani and S. pelagonicus. For example, Salmo duhani is distinguished from S. pelagonicus by having fewer lateral-line scales (115-121, vs. 109-115), a shorter maxilla in males (8—10% SL, vs. 10-11) anda slenderer body in males (body depth at dorsal-fin ori- gin 20-23% SL, vs. 23-27). Taking all into account; the distance, geographic barriers between Macedonia and Ayazma stream, Turkiye and our molecular data; here, we treated this species as S. duhani. Furthermore, bases on our present results, Salmo duhani needs to be redi- agnosed by considering all samples (Génen ve Ayazma streams) in future studies. In the present study, 187,385 unlinked SNP loci shared among the populations were analysed to provide support our recognition of a distinct species. Results provided ev- idence that S. brunoi sp. nov. separates from other Salmo species that inhabit adjacent basins (Figs 5, 6). Zoosyst. Evol. 100 (1) 2024, 87-99 0.8 Ancestry 0.6 0.4 0.2 Adriatic - AD-1 Adriatic - AD-2 Adriatic — AD-3. Atlantic — AT-1 Atlantic — AT-3 Atlantic — AT-4 S. labrax - DA-1 S. ischehan - DA-2 S. ischchan - DA-3 S.marmaratus — MA-2 S.marmaratus — MA-5 S.obtusirostris — OBT-1 97 S. duhani S. duhani S. duhani Salmo_brunoi Salmo_brunoi 5. coruhensis s. coruhensis. s. coruhensis 5. coruhensis S. duhani S. duhani S. duhani S.obtusirosiris — OBT-2 Figure 5. Bar plots of the individuals ancestry generated by ADMIXTURE v.1.3.0 using 187,385 unlinked SNPs. Vertical lines represent each individual and color-code defines the ancestry origin with k= 9 groups. Reference trout specimens are: Adriatic (AD), Atlantic (AT) and Danubian (DA) lineage (originates from two different locations), Salmo marmaratus and S. obtusirostris. Marmara Aegean Basin 0.6 Principal component 2: 21.39% -0.2 © Salmo brunoi @ Salmo duhani © Salmo coruhensis Salmo labrax Salmo ischchan 0.0 Principal component 1: 29.31% Figure 6. Principal Component Analysis (PCA) plot using 187,385 unlinked SNPs. Rectangular symbols represent the Danubian (DA) lineage reference, whereas circles indicate trout specimens used in the study. Colors represent the Salmo coruhensis and S. duhani specimens. Author Contributions DT conceptualized and conceived the idea. EB carried out morphometric measurements under the guidance of DT. SA carried out the fieldwork. MO performed genetic wet- lab work and data analysis and provided funding acqui- sition. The draft was written primarily by DT and all au- thors have read, edited and agreed with the final version. Acknowledgements This work was financially supported by the Scientific Re- search Project Units of Recep Tayyip Erdogan University (Project ID: FBA-2022-1355). MO has received a post- doctoral research grant from The Scientific and Technical Research Council of Turkiye, TUBITAK-BIDEB-2219, at ISEM (University of Montpellier, France). Supplemen- tary funding by the OSU OREME (Univ. Montpellier) for sequencing was appreciated. The wet lab work of the present study was carried out at the GenSeq facility of the University of Monpellier (https://www.labex-cemeb. org/en/genotyping-sequencing-genseq) and sequencing at the MGX platforms (Montpellier, France), respective- ly. GenSeq is supported by the Laboratoire d’ Excellence (LabEx) CeMEB and by ANR “Investissements d’Ave- nir’ program (ANR-10-LABX-04-01). MGX acknowl- edges financial support from the France Génomique National infrastructure, funded as part of ANR “Inves- tissement d’Avenir” (ANR-10-INBS-09). The bioin- formatic analysis benefited from support by K. Belkhir (MBB), M. Leitwein and E. Delpuech from the LDgenX. zse.pensoft.net 98 Therefore, the authors would like to express their sincere appreciation to ISEM and especially to the ‘Biodiversité et Evolution Marine’ team for enabling access to labora- tories to conduct the research. Additionally, we thank J. Freyhof for the specimen loan from FSJF, Fischsammlu- ng, Berlin. 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