Barbatula fluvicola, Calegari & Freyhof & Waldock & Wegscheider & Josi & Rüber & Seehausen, 2025

Calegari, Bárbara B., Freyhof, Jörg, Waldock, Conor, Wegscheider, Bernhard, Josi, Dario, Rüber, Lukas & Seehausen, Ole, 2025, Two new species of stone loaches of the genus Barbatula (Cypriniformes: Nemacheilidae) from Europe with a neotype designation of B. barbatula (Teleostei: Nemacheilidae), Journal of Fish Biology 107 (4), pp. 1364-1397 : 8-17

Barbatula fluvicola , new species

(Figures 4 and 5)

urn:lsid:zoobank.org:act:5AB6B45C-C3BD-409B-A927-D564515 4D2AB

Barbatula sp. ‘Lineage II’ – Alexander & Seehausen 2021:110 – 111 [listed].

Holotype. NMBE 1105500 , 83.8 mm SL, female, Glane River, appr. 900 m upstream of the confluence between Glane and Saane rivers, Aare catchment, Rhine drainage , Canton of Fribourg, Switzerland, 590 m alt., 46 Ǫ 4701.500 N, 7 Ǫ 705.700 E, 5 Oct 2022, Wegscheider, B., Josi, D., Waldock, C. & Canton of Fribourg (Figure 6a) .

Paratype. All from Switzerland: Rhine drainage: Aare River : Canton of Fribourg: NMBE 1105486 – 1105489 , NMBE 1105493 – 1105497 , NMBE 1105499 , 10, 51.2 – 91.2 mm SL (HRXCT of NMBE 1105486), ZFMK-ICH 135303 – 135304 , 2, 81.0 – 81.2 mm SL, ZSM 49670 , 2, 65.4 – 67.3 mm SL, collected with the holotype . Canton of Bern: — NMBE 1105472 – 1105485 , 14 + 1 C&S, 54.0 – 76.7 mm SL, Lyssbach River, at bridge 500 m upstream of confluence with Alte Aare , 440 m alt., 47 Ǫ 5028.100 N, 7 Ǫ 18040.800 E, 18 Oct 2022, Wegscheider, B., Josi, D., Waldock, C, Moreau, S. — NMBE 1105339 – 1105342 , 4, 73.7 – 82.4 mm SL, Limpach River, approx. 1.7 km upstream from Sout-West Golf Limpach on road from Unterramsern to Limpach , 466 m alt., 47 Ǫ 702.800 N, 7 Ǫ 29034.300 E, 27 Jan 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. — NMBE 1105350 , NMBE 1105353 – 1105358 , NMBE 1105360 – 1105361 , NMBE 1105363 – 1105365 , 10 + 4 C&S, 64.8 – 87.3 mm SL, ZFMK-ICH 135305 – 135306 , 2, 76.6 – 80.6 mm SL, NMW 101168 , 2, 78.7 – 81.2 mm SL, Limpach River, 50 m upstream from the bridge on the road Bernstrasse from Wengi to Schnottwil , 471 m alt., 47 Ǫ 5027.300 N, 7 Ǫ 23059.200 E, 23 Feb 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. — NMBE 1105368 – 1105369 , 2, 72.9 – 81.6 mm SL, Limpach River, at Bätterkinden 1 km upstream from the confluence with Emme River , 411 m alt., 47 Ǫ 901000 N, 7 Ǫ 32019.100 E, 2 Mar 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. — NMBE 1111089 – 1111090 , NMBE 1111848 – 1111849 , 4, 84.2 – 87.5 mm SL, Urtene River, village Schalunen approx. 85 m upstream where Alpstrasse street cross the river , 486 m alt., 47 Ǫ 6045.100 N, 7 Ǫ 31053.100 E, 1 Mar 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. — NMBE 1105370 – 1,105,378, NMBE 1105380 , 10, NMBE 1105383 (1 C&S), 66.6 – 86.4 mm SL, NMB 6480 – 6481 , 2, 72.0 – 72.7 mm SL, MNHN 2024 – 1384 , 2, 69.3 – 71.3 mm SL, Emme River, at Wiler bei Utzenstorf next to the Emme Forstbaumschule , 478 m alt., 47 Ǫ 9054.400 N, 7 Ǫ 33018.100 E, 2 Mar 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. Canton of Solothurn: — NMBE 1105385 – 1105387 , NMBE 1105389 – 1105391 , NMBE 1105393 – 1105394 , 8, 54.4 – 71.8 mm SL, MNCN-ICTIO 298.402 and MNCN-ICTIO 298.403 , 2, 67.1 – 69.5 mm SL, Emme River, 1.7 km downstream of the dam of Biberist between Biberist and Derendingen , 486 m alt., 47 Ǫ 11033.900 N, 7 Ǫ 34048.700 E, 4 Mar 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. Canton of Vaud: — NMBE 1105424 , NMBE 1105426 – 1105433 , 11, 39.8 – 68.3 mm SL, ZFMK-ICH 135307 – 135308 , 2, 53.6 – 69.9 mm SL, Petite Glane River, 1 km east from Villars-legrand , 430 m alt., 46 Ǫ 54022.900 N, 7 Ǫ 0027.700 E, 9 Sep 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C., Sudasinghe, H.

Genseq-2 coI. NMBE 1105371 (tissue tag 230097); Genbank Acession Number PQ488083. Genseq-2 coI. NMBE 1105372 (tissue tag 230098); Genbank Acession Number PQ488084. Genseq-2 coI. NMBE 1105487 (tissue tag 240565); Genbank Acession Number PQ488093. Genseq-2 coI. ZFMK-ICH 135303 (tissue tag 240568); Genbank Acession Number PQ488094. Genseq-2 coI. NMBE 1105472 (tissue tag 241024); Genbank Acession Number PQ488169. Genseq-2 coI. NMBE 1111089 (tissue tag 229970); Genbank Acession Number PQ488191. Genseq-2 coI. NMBE 1111090 (tissue tag 229971); Genbank Acession Number PQ488192. Genseq-2 coI. NMBE 1105424 (tissue tag 240249); Genbank Acession Number PQ488148. Genseq-2 coI. NMBE 1105426 (tissue tag 240251); Genbank Acession Number PQ488149. Genseq-2 coI. NMBE 1105340 (tissue tag 229770); Genbank Acession Number PQ488130. Genseq-2 coI. NMW 101168 (tissue tag 229857); Genbank Acession Number PQ488131. Genseq-2 coI. NMW 101168 (tissue tag 229858); Genbank Acession Number PQ488132. Genseq-2 coI. NMBE 1105369 (tissue tag 230083); Genbank Acession Number PQ488133.

Additional material (non-types). Switzerland: Rhine drainage: Canton of Aargau: NMBE 1085028 – 1085033, 6, 62.1 – 79.0 mm SL, Aabach River, tributary of the Aare River, Niederlenz, 363 m alt., 47 Ǫ 24030.100 N, 8 Ǫ 10010.100 E, 14 Sep 2015, NAWA (National Surface Water Quality Monitoring Program; Switzerland). — NMBE 1085259 – 1085272, 14, 35.5 – 84.8 mm SL, NMBE 1106080 – 1106085, 6 (entire vouchers preserved in tissue collection), 35.0 – 39.0 mm SL, Surb River, tributary of the Aare River, Döttingen, 333 m alt., 47 Ǫ 33052.200 N, 8 Ǫ 15052.600 E, 16 Sep 2015, NAWA. — NMBE 1085103, 1, 70.6 mm SL, Bünz River, tributary of the Aare drainage, Möriken, 374 m alt., 47 Ǫ 24035.600 N, 8 Ǫ 11010.100 E, 15 Sep 2015, NAWA. Canton of Appenzell: — NMBE 1083178, tissue (identified by photo), Urnäsch River, Matzingen, 589 m alt., 47 Ǫ 2404.400 N, 9 Ǫ 19034.800 E, 12 Aug 2015, NAWA. Canton of Basel: — NMBE 1085464 – 1085481, 17, 41.6 – 71.0 mm SL, Birs River, Basel, 249 m alt., 47 Ǫ 33024.200 N, 7 Ǫ 3703.800 E, 24 Sep 2015, NAWA. Canton of Bern: — NMBE 1105343 – 1105349, 7, 36.6 – 53.2 mm SL, Limpach River, 1.7 km upstream from Sout-West Golf Limpach on road direction Unterramsern to Limpach, board between Solothurn and Bern cantons, 466 m alt., 47 Ǫ 702.800 N, 7 Ǫ 29034.300 E, 27 Jan 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. — NMBE 1105395 – 1,105,401, NMBE 1105403 – 1105406, 11 + 1 C&S, 64.4 – 92.4 mm SL, Kalte Sense River, tributary of the Aare River, downstream of the Hoflanderebrügg, 957 m alt., 46 Ǫ 42059.200 N, 7 Ǫ 19040.700 E, 6 Sep 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C., Sudasinghe, H., Canton of Bern and Canton of Fribourg. — NMBE 1105407 – 1105422, 16, 39.1 – 83.1 mm SL, Sense River, at Sodbachbrügg, 702 m alt., 46 Ǫ 49036.100 N, 7 Ǫ 19020.700 E, 7 Sep 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C., Sudasinghe, H., Canton of Bern and Canton of Fribourg. — NMBE 1105501 – 1105515, 15, 57.6 – 76.2 mm SL, Schwarzwasser River, tributary of the Aare River, near to Rossgraben bridge, 681 m alt., 46 Ǫ 49037.400 N, 7 Ǫ 23055.500 E, 5 Sep 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C., Sudasinghe, H., Canton of Bern and Canton of Fribourg. — NMBE 1083400 – 1083425, 25, 59.2 – 77.9 mm SL, Gürbe River, tributary of the Aare drainage, Kehrsatz, 507 m alt., 46 Ǫ 550000 N, 7 Ǫ 29029.400 E, 21 Aug 2015, NAWA. — NMBE 1080648, NMBE 1080650 – 1080651, 3, 78.9 – 91.7 mm SL, Schwarzwasser River, tributary of the Aare River, Schwarzwasserbrücke, 587 m alt., 46 Ǫ 51051.500 N, 7 Ǫ 24030.900 E, 17 Sep 2013, unknown collector. — NMBE 1080626, 1, 90.3 mm SL, main course of Schwarzwasser River, at Rüschegggraben, 724 m alt., 46 Ǫ 48011.700 N, 7 Ǫ 24030.900 E, 17 Sep 2013, unknown collector. — NMBE 1080692, 1, 80.0 mm SL, NMBE 1080693 (only tissue), Sense River, tributary of the Aare River, Guggersbachbrücke, 763 m alt., 46 Ǫ 45024.500 N, 7 Ǫ 1801300 E, 16 Sep 2013, unknown collector. — NMBE 1080749 – 1080750, 2, 35.7 – 45.7 mm SL, NMBE 1080751 (only tissue), Sense River, tributary of the Aare River, Ruchmühlebrücke, 609 m alt., 46 Ǫ 50046.700 N, 7 Ǫ 20013.900 E, 17 Sep 2013, unknown collector. Canton of Fribourg: — NMBE 1105449 – 1105454, 5, 46.9 – 89.8 mm SL, Saane River, tributary of the Aare River, 0.6 km upstream of the confluence between Saane and La Gérine, 584 m alt., 46 Ǫ 4603200 N, 7 Ǫ 702000 E, 16 Sep 2022, Wegscheider, B.; Josi, D.; Sudasinghe, H.; Talbi, M., Canton of Fribourg. — NMBE 1080702 – 1080703, 2, 70.8 – 77.7 mm SL, Sense River, tributary of the Aare River, Guggersbachbrücke, 764 m alt., 46 Ǫ 45024.800 N, 7 Ǫ 18010.500 E, 16 Sep 2013, unknown collector. — NMBE 1083482 – 1083483, 2, 69.4 – 89.1 mm SL, NMBE 1106139 – 1106140, 2 (entire vouchers preserved in tissue collection), 43.0 – 44.0 mm SL, Sionge River, tributary of the Aare River, Vuippens, 690 m alt., 46 Ǫ 39022.900 N, 7 Ǫ 4041.800 E, 28 Aug 2015, NAWA. Canton of St. Gallen: — NMBE 1105434 – 1105448, 14 + 1 C&S, 53.7 – 84.8 mm SL, Jona River, tributary of the Limmat River, upstream of Rapperswil-Jona at the bridge, 422 m alt., 47 Ǫ 14012.600 N, 8 Ǫ 50012.800 E, 13 Sep 2022, Calegari, B. B., Wegscheider, B.; Josi, D., Sudasinghe, H. — NMBE 1111834 – 1111837, NMBE 1111057 – 1111067, and NMBE 1109967 – 1109971, 19 + 1 C&S, 64.4 – 81.4 mm SL, Thur River, tributary of the Aare River, upstream of Thur bridge near to Niederbüren, 705 m alt., 47 Ǫ 28011.500 N, 9 Ǫ 11037.900 E, 14 Oct 2023, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. — NMBE 1084866 – 1084886, 19 + 2 C&S, 43.3 – 76.5 mm SL, Thur River, Golfplatz, 476 m alt., 47 Ǫ 2801300 N, 9 Ǫ 11041.700 E, 9 Sep 2015, NAWA. — NMBE 1085366 – 1085367, NMBE 1085369 – 1085374, NMBE 1085376 – 1085377, NMBE 1085380 – 1085382, NMBE 1085384 – 1085389, 19, 57.1 – 82.2 mm SL, Glatt River, Buechental, 496 m alt., 47 Ǫ 26038.400 N, 9 Ǫ 901700 E, 22 Sep 2015, NAWA. Canton of Schwyz: — NMBE 1105455 – 1105470, 16, 63.3 – 100.5 mm SL, Chli-Aa River, tributary of the Limmat River, at Lachen about 400 m upstream of confluence to Lake Zurich, 409 m alt., 47 Ǫ 11052.600 N, 8 Ǫ 51032.500 E, 14 Oct 2022, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C., Schmid, A., Moreau, S. Canton of Thurgau: — NMBE 1083709 – 1083710, NMBE 1083715 – 1083722, 10, 71.0 – 85.0 mm SL, Lauche River, Kubel, 441 m alt., 47 Ǫ 31011.900 N, 8 Ǫ 55050.400 E, 26 Aug 2015, NAWA. — NMBE 1083628 – 1083631, 4, 40.6 – 50.5 mm SL, Chemibach River, Märstetten, 419 m alt., 47 Ǫ 35050.700 N, 9 Ǫ 302000 E, 25 Aug 2015, NAWA. Canton of Zürich: — NMBE 1105531 – 1105545, 15, 57.8 – 77.9 mm SL, Sihl River, tributary of the Limmat River, downstream of bridge near to the train station of Leimbach, 435 m alt., 47 Ǫ 2000.700 N, 8 Ǫ 31011.500 E, 12 Sep 2022, Calegari, B. B., Wegscheider, B., Josi, D. — NMBE 1105546 – 1105547, 2, 85 – 88.3 mm SL, Sihl River, tributary of the Limmat River, downstream of hydroelectric dam Sihlwehr, 705 m alt., 47 Ǫ 10017.700 N, 8 Ǫ 39051.900 E, 12 Sep 2022, Calegari, B. B., Wegscheider, B., Josi, D. — NMBE 1084662 – 1084678, 16, 49.6 – 77.8 mm SL, Mönchaltorfer Aa River, Mönchaltorf, 441 m alt., 47 Ǫ 18043.900 N, 8 Ǫ 43010.500 E, 1 Sep 2015, NAWA. — NMBE 1084301 – 1084307, 7, 47.5 – 67.1 mm SL, Ellikerbach River, downstream at wastewater treatment plant Ellikon, 377 m alt., 47 Ǫ 34020.500 N, 8 Ǫ 49013.400 E, 15 Oct 2015, NAWA. — NMBE 1084369 – 1084383 and NMBE 1106708 – 1106709, 17, 57.1 – 91.8 mm SL, Eulach River, downstream at ARA Elgg, 497 m alt., 47 Ǫ 3004.100 N, 8 Ǫ 5103.200 E, 16 Oct 2015, NAWA. — NMBE 1083144 and NMBE 1083147, 2, 62.6 – 71.0 mm SL, and NMBE 1083143 (only tissue), Furtbach River, tributary of the Limmat River, Otelfingen, 419 m alt., 47 Ǫ 26058.600 N, 8 Ǫ 23012.900 E, 11 Aug 2015, NAWA. Germany: Danube drainage: Bavaria State: — ZSM 35660, 14, 62.8 – 81.9 mm SL, stream Kessel, at Bergmühle, Unterbissingen, West to Donauwörth, Swabia, 48 Ǫ 42028.6200 N, 10 Ǫ 3806.7500 E, 11 Jun 2007, Schubert, M. — ZSM 34592, 20, 80.2 – 46.9 mm SL, stream tributary of the Isar River, Pförreraugraben at Freising, Upper Bavaria, 48 Ǫ 210 N, 11 Ǫ 440 E, 17 Jun 2006, Miller, M. — ZSM 33502, 12, 80.7 – 64.9 mm SL, stream Geltnach, tributary of the Wertach River, below Bertoldshofen near to Marktoberdorf, Upper Bavaria, 47 Ǫ 47.50 N, 10 Ǫ 39.40 E, 6 Jul 2005, A. Kolbinger and B. Ott. — ZSM 32918, 1, 78.9 mm SL, Swabia, 48 Ǫ 19040.500 N, 11 Ǫ 007.800 E, 13 Apr 2005, Schubert, M. — ZMS 31550, 1, 50.6 mm SL, stream Westliche Günz, tributary of the Günz River, upstream of Westerheim near Memmingen, Allgäu region, Swabia, 47 Ǫ 59.70 N, 10 Ǫ 17.50 E, 17 Nov 2004, Schubert, M. and Neumann, D. — ZSM 31620, 2, 57.0 – 58.1 mm SL, Grosse Vils upstream of Lebertshausen, Lower Bavaria, 48 Ǫ 28.80 N, 12 Ǫ 24.10 E, 6 Dec 2004, Kolbinger, A., and Ott, B. — ZSM 33459, 1, 56.0 mm SL, Danube River, upstream on left bank below Danube-bridge at public bath, Dillingen, Swabia, 48 Ǫ 34.30 N, 10 Ǫ 30.80 E, 7 Jun 2005, Schubert, M. — ZSM 37270, 5, 58.9 – 76.9 mm SL, Iller River, 5.6 km South of Ulm, Wiblingen, Swabia,48 Ǫ 21030.200 N, 9 Ǫ 59052.600 E, 23 Jun 2008, Langenargen, F. F. S. — ZSM 36024, 3, 65.0 – 80.0 mm SL, Inn River, at Simbach on the borders between German and Austria, Lower Bavaria, 48 Ǫ 15045.300 N, 13 Ǫ 02011.500 E, 31 Jul 2007, Ott, B., and Bohl, E. Austria: Danube drainage: — NMW 92845, 1, 84.3 mm SL, Fischerwirtsbach, Liefering, Salzburg, 21 Jul 1993, Glechner. — NMW 93066, 4, 26.4 – 64.2 mm SL, Statzenbach, Neumarkt at Wallersee, Salzburg, 28 Jul 1995, Glechner.

Diagnosis. Barbatula fluvicola is a member of a monophyletic group of species, herein designated as the Western Europe clade (including B. barbatula , B. hispanica , B. leoparda and B. ommata n. sp.), which is diagnosed by the following combination of characters. Barbatula fluvicola differs from B. leoparda and B. barbatula by having a shallower head 46.1% – 51.7% HL (vs. 51.5% – 58.3% HL in B. leoparda and 51.7 – 57.3% HL in B. barbatula ), and smaller suborbital depth 26.5% – 32.4% HL (vs. 32.2% – 36.3% and 33.1 – 36.3% HL, respectively). It is further distinguished from B. leoparda and B. hispanica by possessing lower caudal-peduncle depth 8.7% – 11.8% SL (vs. 12.1% – 13.6% and 12.1 – 13.9% SL, respectively); from B. leoparda by having upper lip covering entirely premaxilla (vs. premaxilla visible at mid-line portion), ventral surface of head and area between pectoral to pelvic-fin origin without pigmentation (vs. ventral surface of such areas pigmented with brown to dark irregular blotches), a shorter posterior nareal distance 21.3 – 26.5% HL (vs. 26.8% – 30% HL), and a shorter lower lip 13.7 – 17.2% HL (vs. 17.4% – 21.2% HL), and from B. hispanica by having a shorter maxillary barbel, reaching to middle of eye (vs. long maxillary barbel, reaching to posterior border of eye). It is further distinguished from B. barbatula by having a shorter dorsal head length 18.4% – 21.7% SL (vs. 23.2% – 25.8% SL), a smaller dorsal-fin base length 11.2% – 13.6% SL (vs. 14.5% – 18.6% SL), slope preceding anterior nares absent or weakly developed (vs. protuberant slope present from preceding area of anterior nares) and adpressed anal-fin tip reaching about middle of caudal peduncle (vs. adpressed anal-fin tip slightly anterior to end of caudal peduncle). Additionally, the new species distinguished from the sympatric congener B. ommata n. sp. by having smaller orbit 16.1% – 19.8% HL (vs. 21.1% – 28.3% HL), deeper head 46.1% – 51.7% HL (vs. 38.4 – 46% HL), a longer coracoid, occupying at least two thirds of cleithrum length, usually almost reaching its tip (vs. coracoid short, half-length of cleithrum; Figure 7), ventral border of dentary curved (vs. ventral border of dentary straight; Figure 8), a smaller swimbladder bony capsule, not reaching half of head depth (Figure 9) (vs. swimbladder bony capsule large, occupying slightly more than half of head depth), posterior border of swimbladder bony capsule straight in ventral view (Figure 9) (vs. posterior border of swimbladder bony capsule rounded) and condyle of quadrate positioned at vertical line passing through posterior naris (Figure 9) (vs. condyle of quadrate positioned at vertical passing through anterior border of orbit).

Description. Morphometric data are provided in Table 2. Body robust elongate, roughly cylindrical in cross-section at trunk, gradually compressed towards caudal peduncle. Dorsal profile straight to slightly convex from snout tip to dorsal-fin origin. Dorsal and ventral profile of trunk approximately straight. Dorsal head profile slightly concave from snout tip to anterior border of eye and relatively straight from that point to posterior border of supraoccipital. Some individuals with shallow or conspicuous hump demarking posterior border of supraoccipital. Body depth decreasing posterior to dorsal-fin base to middle caudal-fin peduncle. Greatest body depth slightly anterior to dorsal-fin origin or at half distance between pectoral-and pelvic-fin origins at abdominal area. Greatest body width in half distance between pectoral-and pelvic-fin origins.

Head relatively deep, cylindrical in dorsal view, longer than wide. Eyes laterodorsally positioned on dorsal half of head; located at middle between snout tip and posterior opercle margin. Ocular capsule slightly ellipsoid formed by thin and transparent skin. Nostrils small; anterior naris opening slightly smaller than posterior one. Anterior nostril with an elongate fleshy flap-like tube. Posterior naris in a drop shape. Anterior and posterior nostril close to each other, connected by a thin skin ridge.

Ventral border of gill opening attached to body at inner pectoral-fin base. Mouth ventral, arched in a semicircular shape. Mouth wide, almost equal to head width. Upper lip thick, covering all premaxilla area. Mid-portion of upper lip larger than lateral portion. Short median incision (notch) in upper lip reaching to half lip depth; poorly developed in some individuals. Upper lip papillose in some individuals, smooth in others. Anterolateral portion of upper lip more depressed than middle portion. Outer skin of upper lip with three protuberances behind interspace of barbels. Lower lip separately at midline by a deep notch. Mesial portion of lower lip well-developed into a mental lobe vertically elongated formed by a fleshy fold in inverted U-shaped. Mental lobes usually in close contact at mid-line; without protrusion, leaving just a small V-shaped space of dentary uncovered at mid portion. Lateral portion of lower lip positioned inclined in 45 Ǫ angle to ventral midline, with deep furrows, posteriormost tip without projection.

Distal parts of furrows on lower lip with small papillae, proximal parts usually smooth. Tip of mesial premaxillary barbel reaching approximately to base of maxillary barbel. Lateral premaxillary barbel reaching anterior border of posterior naris, when folded upwards. Maxillary barbel reaching or slightly surpassing vertical of anterior margin of eye, never surpassing eye.

Pectoral-fin rays i,10 (15), i,11* (17) or i,12(4). Innermost ray unbranched and very small, usually visible only in C&S specimens, with a single process base attached to first radial. Distal margin of pectoral fin truncated or convex. Principal unbranched pectoral-fin ray two thirds of longest pectoral-fin ray. Pelvic fin rays i,6 (3) or i,7* (33), plus one additional, tiny, unbranched ray (sometimes absent). Pelvic fin ellipsoid, its origin at vertical through dorsal-fin origin or at principal, unbranched dorsal-fin ray. Pelvic fin with distal margin convex, its tip slightly anterior to a point below end of adpressed dorsal fin. Axillary pelvic lobe present, conspicuous fully attached to body. Dorsal-fin origin situated approximately at middle of body, straight or slightly convex. Proximal radial component of first dorsal-fin pterygiophore inserted anteriorly to neural spine of 13th vertebrae (11 C&S). Dorsal fin with 3 – 4 small (11 C&S), unbranched rays completely covered by skin, followed by i,7½* (30) or i,8½ rays (6). Dorsal fin with 8 (10 C&S) to 9 (1 C&S) pterygiophores and one additional posterior smaller pterygiophore with its radial element (sometimes radial absent) not bearing any rays. First dorsal-fin pterygiophore composed of likely supraneural and first proximal radials fused to each other in single complex structure supporting 3 – 4 shorter unbranched rays + principal unbranched ray. Remaining pterygiophores bearing posterior laminar bony keel supporting 7 – 8½ branched rays, each one associated individually to one of those pterygiophores, last 1½ rays supported by penultimate pterygiophore, and one additional last pterygiophore smaller in size lacking any correspondent ray. Dorsal-fin principal unbranched ray shorter than subsequent posteriormost four branched rays, reaching two thirds of its length. Distal anal-fin margin straight and slightly inclined anteriorly. Anal fin with three simple rays plus i,5½ *(36) rays (one specimen with first two principal rays unbranched; morphological aberration). First simple rays and first unbranched principal ray supported by composed structure of at least three pterygiophores fused into single element, and five pterygiophores supporting remaining branched rays and last radial element much smaller at end of fin without rays attached; ultimate pterygiophore supporting 1½ rays. First pterygiophore inserted anteriorly to hemal spine of 24th or 25th vertebrae (11 C&S). Caudal fin truncated or slightly emarginate. Upper and lower lobe of caudal fin equal in length. Caudal-fin rays i,8 + 7,i* (18), i,7 + 8,i (5), i,8 + 8,i (12), or i,9 + 8,i (1 C&S). Procurrent rays in dorsal lobe 7 – 10, and ventral lobe with 7 rays. Procurrent rays pronounced and embedded in skin of shallow dorsal and ventral adipose keels extended on at least half of caudal peduncle. Ventral adipose keel slightly elevated extending to tip of adpressed anal fin. Epural long articulated to base of vertebrae. Upper hypural plate as single element with fused hypurals 3, 4 and 5. Lower hypural plate with co-ossified parhypural and hypurals 1 and 2 separated, forming the compound caudal centrum. Total vertebrae, 39 (8 C&S) or 40 (3 C&S); 14 – 15 (11 C&S) pairs of ribs.

Body covered by embedded, tiny, rounded, not overlapping scales, irregularly set along trunk. Scales present on flank from about middle of pectoral fin to caudal-fin base. No scales on pre-dorsal region, except small patch before dorsal-fin origin. Scales on ventral surface of body present from end of pelvic-fin girdle to urogenital opening. Dermal tubercles present in some individuals, dense on head in adult males and females, and on dorsal surface of pectoral-fin rays.

Lateral line continuous and complete, reaching caudal-fin base, with 64 – 67 pores (11 C&S), first three pores visibly larger than remaining. Cephalic lateral sensorial system with canals only partially ossified. Infraorbital canal with 11 – 12 pores (formed by two pores + posterior-half pore of antorbital segment, and anterior-half pore + nine pores + posterior-half pore of suborbital segment). Infraorbital canal composed of antorbital branch bearing two pores (anterior and dorsomedial), posteriorly ending in half-pore close but separately from anterior-half pore of adjacent suborbital branch, followed by nine pores and ending in posterior-half pore attached to distinct postorbital canal. Postorbital canal turned backwards with anterior-half pore, plus three pores and posterior-half pore in its end connected to both supratemporal canal and lateral line canal (or trunk canal). Supratemporal canal with half pore connected to both posterior-half pore of postorbital and half pore of lateral line canal, and two pores dorsally positioned. Trunk canal with three larger pores. Nasal canal with three pores weakly ossified (anterior, lateral and posterior). Frontal canal reaching near to anterior orbital border, but truncated at mid-portion splitting in two separate branches, each one bearing three pores (anterior, lateral and posterior). Preopercular canal with eight pores, anterior part located ventrally to articular and posteriorly to dentary.

Colouration (in alcohol). Background colouration yellowish, head, back and flank with dark-brown pattern (Figure 10). Head very densely mottled. Back and flank mottled or marmorated, body and head almost plain brown in some individuals. Juveniles smaller than 40 mm SL often with inconspicuous, irregular shaped, blotches on back, most constant below dorsal-fin origin and at end of dorsal-fin base. Inconspicuous series of blotches along lateral midline in some individuals. In individuals with plain-brown flank, pigmentation dissociating and fading, below a line between origin of pectoral and pelvic fins and abdominal cavity. Ventral portion of head and abdomen area without pigmentation. Lateral part of head with dense pigmentation on cheek, lacking melanophores on ventral portion of the opercle. Presence of thin stripe from snout tip to anterior portion of eye, limited dorsally by nasal opening. Pigmentation absent on maxillary barbel, except when present, only on base. Premaxillary barbel usually without pigmentation, but when present more frequently in lateral pair of barbel with irregular dark markings on dorsal surface, most concentrated on proximal half. Lips completely deprived of pigmentation except by anterior border of upper lips. Pectoral fin with elongate dark blotches on rays, distal margin unpigmented. Blotches larger, more concentrate and conspicuous along anterior half of fin (first six rays). Dorsal-fin rays with small, somewhat rectangular dark blotches on rays, distributed irregularly, but sometimes organised in three bands, distal dorsal fin border without pigmentation. Pelvic fin hyaline. Anal fin hyaline, in few individuals with a faint vertical row of pigmentation on base. Caudal fin with small, elongated blotches on rays, forming three to four bands in most individuals, without bands in others. Distal margin of caudal fin unpigmented.

Colouration in life. Similar to that observed in preserved specimens, but with fins and maxillary barbels orangish yellow to orange, more evident in paired fins.

Remarks (variability). All specimens identified as Barbatula fluvicola were readily identifiable by their overall body and head shape and proportions. In some geographically restricted areas, phenotypes of some individuals did not match the species assignment based on genetic barcode (see in Molecular analyses and species delimitation Section). This was the case in the streams Jona and Chli-Aa, both in the Limmat drainage (listed as non-type material), near to the confluence with Lake Zürich, where a few individuals are intermediate in phenotype and colour pattern between B. fluvicola and B. ommata n. sp., suggesting hybridisation in a secondary contact zone. Two individuals from Lake Zürich, near to the inflow of the stream Jona (NMBE 1071779 and NMBE 1071775), were molecular assigned to B. fluvicola , corroborating this hypothesis. On the other hand, the loach population of Constance and Geneva lakes recovered as nested in the B. fluvicola clade in coI tree were differentiated in several traits from B. fluvicola , such as having larger snout length and eyes, and a narrower head in adults, but recovered these populations nested. Since we are uncertain if these populations from the Constance and Geneva lakes represent hybrid populations in a contact zone or a distinct species closely related to B. fluvicola , which has recently diverged (see discussion); we are herein eventually assigning those populations as Barbatula aff. fluvicola until further work can resolve their status (see Comparative Material section).

The molecular analysis further assigned part of the population from Schwarzwasser stream in the Sense River catchment (NMBE 1080648, NMBE 1080650, NMBE 1080651), Sense River itself (NMBE 1080702, NMBE 1080703, NMBE 1080692, NMBE 1080750, NMBE 1080751, NMBE 1105471 and NMBE 1105408), Saane River (NMBE 1086859 and NMBE 1105452) and some individuals of Furtbach in the Limmat River catchment (NMBE 1083146, NMBE 1083148, NMBE 1083143, and NMBE 1083144) to B. ommata n. sp. However, these individuals clearly share the overall morphology and diagnostic characters of B. fluvicola , and they are herein assigned as such. We hypothesised an indicative of past introgression from the latter species (see discussion). Further clarification of the status of these populations will require a population genomic and phylogenomic analyses (Calegari et al. in prep.). For now, the few individuals with mismatch between phenotype and mitochondrial haplotype are listed as non-type material under B. fluvicola .

Distribution. Barbatula fluvicola is known from several tributaries of the upper Rhine drainage (here defined to include also high Rhine and Alpine Rhine drainage sections), including rivers in the Aare, Reuss and Limmat catchments. The species is absent from lakes in the Aare catchment. The species is also found in the upper Danube drainage in Germany and in the Inn drainage in Austria (vouchers listed in non-type series were morphologically and genetically confirmed). The overall distribution of the species comprises the upper Rhine drainage in Switzerland and Germany, and upper Danube River drainage in Germany, Austria and Switzerland (Figure 3).

Sexual dimorphism. Epidermical tubercles in pectoral fin present mostly in 1st – 4th branched rays in males (vs. tubercles absent or not evident in females and juveniles). Differences in pectoral-fin shape are easily noticed in adults, with males showing pointed pectoral-fin tip and enlargement of outermost branched-fin rays (vs. rounded in females); juveniles and some subadults showed no conspicuous differences in pectoral fin shape. Urogenital papilla of males relatively small and conical (vs. rounded in females).

Etymology. The name fluvicola is from Latin meaning inhabitant of rivers, alluding to the stream preference habitats where this species has been collected. A noun in apposition.

Vernacular name. North-Prealpine Stone Loach (English), Nord-Voralpine Bartgrundel (German), Loche Préalpes du Nord (French).

Ecological notes. Specimens were collected in small to medium size rivers, clear water, with swiftly flowing water. This species inhabits at the bottom of rivers under stones and among pebbles and sometimes close to larger rocks when in more rapid deeper waters. Juveniles are often found associated with greater water moss ( Fontinalis antipyretica ) and algae, as well as aquatic plants, which seems to be an important habitat providing protection against predators. Water parameter ranges from sampled sites overall all seasons inhabited by B. fluvicola : temperature from 4.8 to 18.2 ǪC; pH between 7.57 and 8.4; conductivity from 350 μs/cm to 640 μs/cm. The species seems to be primarily insectivorous based on few individuals dissected for clearing and staining technique (listed in type material).

Conservation status. Barbatula fluvicola has a wide distribution and it is known in Switzerland from the Aare, Reuss and Limmat drainages, tributaries of the Rhine River, and from the upper Danube and its tributaries in Germany, and the Inn drainage in Switzerland and Austria. The extent of occurrence (EOO) is estimated in 47,420 km 2 by the Minimum Convex Polygon, and the area of occupancy (AOO) is about 188 km 2 (calculated using GeoCAT tool). However, it is expected that the species also occurs in similar habitats along other portions of its distribution in Upper Rhine and Danube drainages in Germany and Austria. Part of the distribution of the species in Switzerland (mainly subpopulations from Aare catchment around Bern and Fribourg cities, and Limmat catchment) is situated in large urban and agricultural areas that is experience impacts on its habitat quality due to urbanisation and pesticides. Additionally, almost all rivers inhabit by the species in Switzerland are fragmented by several artificial small to median barriers; however, this species is not migratory and may not represent a direct impact that could threaten the population long-term persistence. Barbatula fluvicola is relatively widely distributed, and despite it suffering continued negative impacts in its occupancy area due to decreasing habitat quality, those threats do not affect a majority of localities, where the species seems to be abundant. Thus, Barbatula fluvicola is preliminary categorised as Least Concern (LC) according to IUCN criteria (IUCN Standards and Petitions Subcommittee, 2022). However, it is recommended that monitoring of the species subpopulations and its area of occupancy be conducted for better understanding of its abundance and population stability.