Two new species of stone loaches of the genus Barbatula (Cypriniformes: Nemacheilidae) from Europe with a neotype designation of B. barbatula (Teleostei: Nemacheilidae)

REGULAR ARTICLE Two new species of stone loaches of the genus Barbatula (Cypriniformes: Nemacheilidae) from Europe with a neotype designation of B. barbatula (Teleostei: Nemacheilidae) Bárbara B. Calegari 1,2,3 | Jörg Freyhof 4 | Conor Waldock 1,2,3 | Bernhard Wegscheider 1,2,3 | Dario Josi 1,2,3 | Lukas Rüber 1,5 | Ole Seehausen 1,2 1 Aquatic Ecology and Evolution, Institute of Ecology and Evolution, University of Bern, Bern, Switzerland 2 Department of Fish Ecology and Evolution, EAWAG, Swiss Federal Institute for Aquatic Science and Technology, Kastanienbaum, Switzerland 3 Wyss Academy for Nature at the University of Bern, Bern, Switzerland 4 Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Berlin, Germany 5 Naturhistorisches Museum Bern, Bern, Switzerland Correspondence Bárbara B. Calegari, Aquatic Ecology and Evolution, Institute of Ecology and Evolution, University of Bern, Bern, Switzerland. Email: [email protected] Funding information Kanton Bern; Wyss Academy for Nature; Swiss Federal Office of the Environmental Abstract Ten species of Barbatula are recognised in Europe, west of the Urals: B. barbatula, B. caucasica,B. hispanica,B. leoparda,B. pironae,B. quignardi,B. sturanyi,B. taurica, B. vardarensis and B. zetensis, with B. caucasica and B. taurica formerly considered subspecies of B. barbatula. A comprehensive dataset of the DNA barcoding gene coI recovered four major clades within Europe: three in Eastern Europe including B. caucasica,B. pironae,B. sturanyi,B. taurica,B. vardarensis and B. zetensis, and one in Western Europe including B. barbatula,B. hispanica and B. leoparda. The results further indicated several genetic lineages, representing potentially new species. Recent surveys in Switzerland revealed two new species of Barbatula, within the Western clade, which are herein described. Barbatula fluvicola, a new species, inhabits streams and rivers in the upper and middle Rhine drainage in Switzerland and Germany, as well as the upper Danube drainage in Germany and Austria. Barbatula ommata, a new species, is mostly confined to lakes of the Aare-Rhine system. The two new species overlap geographically in Switzerland, where they occupy different habitats. Morphological differences, species delimitation analyses, phylogenetic reconstruction and genetic distances based on the coI gene corroborates the recognition of the two new species. To stabilise the nomenclatural status and the consequent use of the nomen B. barbatula, we are herein designating an unambiguously identifiable neotype from the Lez River population, previously recognised as B. quignardi, to clarify the identity of the nominal species Cobitis barbatula Linnaeus, 1758. KEYWORDS biodiversity, Danube River, European fishes, freshwater, lakes, Rhine River, systematics urn:lsid:zoobank.org:pub:DD3166A7-8A96-40C9-86F2-7D060384500C. Received: 27 November 2024 Revised: 16 April 2025 Accepted: 19 May 2025 DOI: 10.1111/jfb.70108 FISH This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2025 The Author(s). Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles. J Fish Biol. 2025;1–34. wileyonlinelibrary.com/journal/jfb 1 1|INTRODUCTION In Europe, nemacheilid loaches comprise two genera, Barbatula Linck, 1790, and Oxynoemacheilus (Banarescu & Nalbant, 1966). Stone loaches of the genus Barbatula are very common throughout Europe and northern Asia, from the Mediterranean basin in Spain to Japan and the eastern parts of Siberia (Kottelat & Freyhof, 2007). Currently, seven valid species are recognised in Western and Central Europe: Barbatula barbatula (Linnaeus, 1758), B.sturanyi (Steindachner, 1892), B.vardarensis (Karaman, 1928), B.quignardi (B acescu-Mes¸ter, 1967), B.hispanica (Lelek, 1987), B.zetensis (Sori c, 2000) and B.leoparda Gauliard et al., 2019. The type species, Barbatula barbatula, was formally described by Linnaeus (1758), who based his account on Linnaeus (1746:125, gen. 2, syn. 2, spec. 332) and Artedi's (1738: gen. II, syn. 2, spec. 2), on which a compilation of previous literature accounts were based (complete literature accounts listed below). During the 20th century, all nominal species listed above, with the exception of B.leoparda, were treated as synonyms of B. barbatula (Kottelat, 1997; Vasil'eva, 1998; Sori c, 2000). However, Kottelat and Freyhof (2007) recognised three of those previously considered synonyms as valid species, B. quignardi,B. sturanyi and B. zetensis. With the increasing availability of DNA data (cytochrome c oxidase subunit I,cytochrome b and the 12S), it became evident, that B. barbatula includes several undescribed species highlighted by the presence of several distinct evolutionary lineages (Barluenga & Meyer, 2005;ˇ Sedivá et al., 2008; Knebelsberger et al., 2015; Norén et al., 2017; Behrmann-Godel et al., 2017; Denys et al., 2021; Zangl et al., 2022; Clavero et al., 2023). Often mistaken as a cryptic species (one or more species classified as a single species due to indistinguishable morphology but distinct genetically; Struck & Cerca, 2019), B.barbatula is instead a species complex lacking sufficient taxonomic study to recognise and formally describe its distinct species. To do so, there is also a need to stabilise the accurate use of the nomen B.barbatula. During the characterisation and inventory of fish diversity of lakes and rivers in Switzerland (Alexander & Seehausen 2021; Brodersen et al., 2023), molecular analyses revealed three major molecular lineages in the genus Barbatula. The aim of this study was to recognise and delimitate the evolutionary lineages in Barbatula occurring in Switzerland and adjacent regions, and formally describe them as species. 2|MATERIALS AND METHODS 2.1 |Samples, comparative material and morphological procedures Individuals used for morphological analyses were collected by the authors as part of the LANAT-3 project (Predicting and Preventing the Biodiversity Loss in Water Bodies under Climate Change) in 2022 and 2023. Samples from Switzerland were collected by electrofishing under the Swiss Animal Experiment permit (National number 34546 and 34150). Once captured, fish were anaesthetised and euthanised using an overconcentrated solution of MS-222 (tricaine methanesulfonate). Photographs of live specimens were taken just after individuals were anaesthetised. Preserved fishes including types as well as tissue samples collected in Switzerland (muscle and fin clip) were catalogued at the ichthyological collection of the Naturhistorisches Museum Bern (NMBE). Specimens were fixed in 10% formaldehyde and posteriorly preserved in 75% ethanol. The specimens examined in this study are those listed in the comparative material section, and institutional abbreviations are those listed at http://www.asih.org/codons.pdf (Sabaj, 2023). The species distribution map of the new species and related valid species was developed using the HydroSHEDS database (hydrological data and maps based on shuttle elevation derivatives; Lehner et al., 2008;Lehner& Grill, 2013;http://www.hydrosheds.org), as well as the elevation DEM shape file prepared in QGis (QGIS Development Team, 2009)version 3.16.14 (2022) following Calegari et al. (2016) and Calegari and Fontanelle (2017). The geographic distribution map of molecular sequences used and plotted in the phylogeny was produced using the R software (Core Team, 2013) package phytools V.2.3-0 (Revell, 2024)with‘phylo. to.map’. In the species account, geographical descriptors (e.g. cantons and city names) are written in the language of the country or canton of origin to avoid translation errors. For simplicity, we use English for lake names. The conservation status of the new species was assessed following the categories and criteria of the International Union for Conservation of Nature (IUCN Standards and Petitions Subcommittee, 2022). Geographical range information for the species was compiled from known material and managed in the GeoCat tool to calculate extent of occurrence (EOO) and area of occupancy (AOO). Morphometric measurements were obtained to the nearest 0.1 mm with digital callipers under a stereomicroscope, preferably on the left side of an adult specimen following Kottelat and Freyhof (2007). Additional measurements included the following: distance between pectoraland pelvic-fin origins, distance between pelvicand anal-fin origins, first dorsal-fin ray length (measured from dorsal-fin origin to posterior tip of first unbranched dorsal-fin ray), first pectoralfin ray length (measured from pectoral-fin origin to posterior tip of first unbranched pectoral-fin ray), first pelvic-fin ray length (measured from pelvic-fin origin to posterior tip of first unbranched pelvic-fin ray), body width at dorsal-fin origin (measured between lateral margins of body at dorsal-fin origin), head depth (measured between dorsal–ventral borders of head at middle of eye), anterior internarial distance (measured horizontally between inner margins of anterior nares), posterior internarial distance (measured horizontally between inner margins of posterior nares), distance between upper and lower lips (measured from midline of upper lip to posterior lateral tip of lower lip), lower-lip length (measured from anterior border of mental lobe to posterior tip of lateral portion of lower lip), mental-lobe length (measured from anterior border to posterior border of mental lobe of lower lip) and mouth width (measured between lateral borders on posterior portion of mouth). Morphometric data are expressed as percentage of standard length (SL), except subunits of the head, which 2CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License are expressed as percentage of head length (HL), here represented by lateral head length to make our measurements comparable to previous studies (Denys et al., 2021; Gauliard et al., 2019). Measurements were taken from the new species and the closest related species (B. barbatula,B.hispanica,B.quignardi and B.leoparda), including, but not restricted to, type series and/or individuals from type locality. Additional morphological information for comparative analyses was taken from Gauliard et al. (2019) and Denys et al. (2021). Meristic variables were counted in ethanol preserved adults and in cleared and counterstained specimens (C&S) for bone and cartilage according to Taylor and Van Dyke (1985). The laterosensory system canals and pores, fin-radials, ribs and vertebrae, together with osteological features were assessed in C&S specimens or computed tomography (CT) scan images. Meristic data of neotype and holotypes are indicated by an asterisk between parenthesis when variation within species is present. The count of vertebral centra includes those in the Weberian complex (first fourth fused vertebrae) and the compound caudal centrum (PU1 +U1) associated with the hypural complex as a single centrum. The posteriormost dorsaland anal-fin soft rays are usually composed of two pterygiophores supported by the same radial. We considered the last two dorsaland anal-fin soft rays as compound rays represented by 1½ when counted. The nomenclature of the laterosensory canals and associated pores follows Rizzato and Bichuette (2016), except for the infraorbital canal series. The infraorbital series in Barbatula is considered herein the composition of the antorbital segmentation (canal branch of lacrimal of Prokofiev, 2010) and the suborbital canal series that are connected to each other. Additionally, we assumed a postorbital canal as a single series in contrast to the division in sphenotic +otic +postotic canals presented by Rizzato and Bichuette (2016) (Figure 1). To avoid any confusion and to make comparison with other literature possible, we also give the infraorbital pore numbers of antorbital and suborbital separately in parentheses alongside the description. The nasal and frontal canals are usually referred as a single canal (supraorbital canal) in Prokofiev (2010), but they represent two independent, distinct canals and we herein count them as independent of each other. The infraorbital canal series in stone loaches is composed of an anterior portion that runs posteriorly parallel to the axis formed by the anterior and posterior nares, identified as the antorbital portion of the infraorbital canal, which lies just below the skin surface, externally in a long-ossified tube bearing an anterior separate branch with pores. The second portion of the infraorbital series is composed of canals running ventrally along the eye border, is weakly ossified and turns dorsally in the end portion in the direction of the supratemporal canal (Figure 1). Morphometric analyses and statistics were performed with the software Past 4.11 (Hammer et al., 2001). Principal component analysis (PCA) was used to assess the overall morphometric variation among populations and species. To reduce the correlation between variance and specimen size, and to ensure morphometric characters were normally distributed, they were log-ratio transformed (Aitchison, 1986), therefore the first principal component that mostly accounts for size variation was maintained. High-resolution X-ray computed tomography (HRXCT) was carried out along the long axis of the specimens of the two new species, from snout tip to about the sixth vertebrae. The specimens were scanned in the facility of the Institute of Anatomy, University of Bern, Switzerland using an SkyScan 2214 under the follow settings: power 60 kV, middle focal spot size X-ray source, current 140 μA, no filter, Hamamatsu detector, 2 stacks, total slices 2205, rotation step 0.1,no frames averaged, 0 skip frames and performed under the following parameters according to the species. Barbatula fluvicola n. sp.: exposure time of 1422 ms, source to object 27.251, source to detector 313.563, 3309 projections, voxel size 0.00650072 mm. Barbatula ommata n. sp.: exposure time of 1482 ms, source to object 23.054, source to detector 313.563, 2818 projections, voxel size 0.0748 mm. Visualisations and sectioning of the three-dimensional models were produced as 8-bit png files with grey values mapped for background set to zero in the software package VGStudio MAXt V1.2.1 by the Institute of Anatomy facility. Segmentation of bones were made in FIGURE 1 Schematic comparison of the laterosensory system of canals of (a) B.fluvicola and B.ommata, and (b) illustration of the nomenclature for canals used in this work. The diagram represents the lateral view of left side of the head. Pores are marked in black, and the ones marked in grey are formed by half-pores of distinct, adjacent canals. An, anterior naris; pn, posterior naris. CALEGARI ET AL.3 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Amiro-Avizo 3D 2023.2, and the meshes were coloured in MeshLab Ink 2023.12, where figures were captured as still frames from HRXCT digital animations for posterior edition in Adobe Photoshop v.2024. 2.2 |Molecular procedures and species delimitation Genseq nomenclature for the new species follows Chakrabarty et al. (2013), and coI sequences from secondary types are listed below the type material of each new species. To assess the genetic diversity within the genus Barbatula, we downloaded 267 coI sequences from GenBank/BOLD. An additional 317 previously unpublished Barbatula coI sequences were obtained from the project FREDIE (Freshwater Diversity Identification for Europe, www.fredie.eu) and finally, 189 coI sequences were specifically generated for this project. Sample metadata are provided in Data S1. DNA extraction, PCR amplification and sequence generation of the FREDIE samples followed Geiger et al. (2014) and those for the remaining samples followed Conte-Grand et al. (2017) using the universal coI fish barcoding primers FishF1 and FishR1 (Ward et al., 2005). Raw reads were edited and assembled into contigs using Geneious Prime v2022.0.2 and individual consensus sequences together with sequences from GenBank/BOLD and FREDIE were aligned using MAFFT v7.017 (Katoh & Standley, 2013), as implemented in Geneious Prime using default options. Triplophysa minxianensis (GenBank accession KT213596) was used as outgroup for the subsequent phylogenetic analysis. PartitionFinder 1.0.1 (Lanfear et al., 2012) was used to assess the optimal partitioning for subsequent maximum likelihood (ML) analyses using the settings (model_selection =BIC search =greedy). ML analyses were conducted with RAxML v7.3.460 (Stamatakis, 2006), implementing the GTRGAMMA model under the -f a setting and 1000 bootstrap replicates. In addition to the dataset composed of all Barbatula sequences from Asia and Europe (all Barbatula dataset, henceforth referred to as AB dataset, consisting of 773 taxa and Triplophysa minxianensis as the root of the tree), we also run independent ML analyses for the European Barbatula only (European Barbatula dataset, henceforth referred to as EB dataset, consisting of 718 ingroup taxa and B.conilobus and B.toni as outgroups) and the Western European Barbatula only (Western Barbatula dataset, henceforth referred to as WB dataset, consisting of 418 ingroup taxa and B.pironae,B.sturanyi and B. zetensis as outgroup). Genetic distances (pdistances) within and between species or clades of interest were calculated in PAUP* v4.0a169 (Swofford, 2002). Molecular operational taxonomic units (MOTUs) were delineated using the single locus species delimitation methods assemble species by automatic partitioning (ASAP, Puillandre et al., 2021) as implemented in the ASAP webserver (https://bioinfo. mnhn.fr/abi/public/asap/), Poisson tree processes (PTP; Zhang et al., 2013; Kapli, Lutteropp, Zhang, Kobert, Pavlidis, Stamatakis, et al., 2017) and multi-rate Poisson tree processes (mPTP, Kapli, Lutteropp, Zhang, Kobert, Pavlidis, Stamatakis, et al., 2017), as implemented in the mPTP webserver (https://mptp.h-its.org) using the following settings mptp -ml -single -pvalue 0.001 and mptp -ml -multi, respectively. For comparison, ASAP, PTP and mPTP based species delimitations were conducted on the EB and WB datasets. 3|RESULTS 3.1 |Nomenclature Barbatula barbatula was described as Cobitis barbatula by Linnaeus (1758: 303), who based his account on Linnaeus (1746: 125, gen.2, syn. 2, spec. 332) and Artedi's (1738: gen. II, syn. 2, spec. 2) description of ‘Cobitis tota glabra maculosa corpore subtereti’. Linnaeus (1746) account is largely identical with the one from Artedi (1738). Linnaeus (1758: 303) gives a general statement in its description ‘C. cirris oris 6, capite inermi compreffo’providing four sets of meristic characters citing his own publication in 1746 as said ‘faun. Svec. Idem’. However, Linnaeus (1746) did not provide any meristic data in this publication, thus whether these counts were originally from his study (1758) or based on other earlier authors is uncertain. Linnaeus (1758) also summarised the known distribution of the species as ‘Habitat in Europae’. Artedi (1738) lists its common names in Germany (Grundel, Gründling, Smerle and Smerling), England (The loche or Groundling) and France (Loche franche), and Linnaeus (1746) mentions its common name in Sweden (Grönling) and Germany (Grundel). However, the species accounts of Linnaeus (1746) and Artedi (1738) were based on several previous bibliograph references (Aldrovandi, 1613; Charletonus & Charleton 1668; Figulus, 1540;Gesner,1563;Jonstonus,1649; Ray, 1713; Rondelet, 1555;Schonevelde,1624;Willughby,1686). Norén et al. (2017) discussed in detail the actual and historical records of stone loaches from Sweden, including individuals potentially seen by Linnaeus in the natural history collection of King Adolf Fredrik I. No specimen was listed or mentioned in the descriptions of Linnaeus (1746,1758) or Artedi (1738), which were only based on ancient literature references. Linnaeus (1746: 125) mentioned that the species inhabiting the Swedish lake Mälaren was brought from Germany by King Fredrik I. Following Norén et al. (2017), B. barbatula was never again reliably reported from lake Mälaren. Norén et al. (2017) stated that it remains unclear if Linnaeus found the species in lake Mälaren or if he just reports on materials seen in the natural history collection of Adolf Fredrik, although a jar catalogued under the number NRM 68 in Adolf Fredrik's collection holds two specimens 72.0–73.2 mm SL that were certainly present in Adolf Fredrik's collection during Linnaeus times and these are subsequently described by Linnaeus (1764). The case of the preserved specimens is complicated, however, because part of the Linnaeus (1764) description of the species derives from another jar, NRM 69, identified as Misgurnus fossilis. NRM 68 might be the only potential surviving syntypes but the uncertainty around its syntype status does not allow to select it as a lectotype. Fernholm and Wheeler (1983) already excluded NRM 68 from the syntype series of B. barbatula but Kottelat (2012) mentioned them as potentially surviving syntypes. At Linnaeus' time, the ‘type’concept did not exist yet, i.e. new species/subspecies were described without lists of type-specimens or even of a mention of the number 4CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License of specimens used for the description (Dubois et al., 2014). In the case of these old publications, the syntypes must be identified a posteriori. According to Article 72.4.1 (International Commission on Zoological Nomenclature, 1999; ICZN) the type series of a nominal speciesgroup taxon consists of all the specimens included by the author in the new nominal taxon (whether directly or by bibliographic reference). In the case of Linnaeus (1758), who based his work on the account of Artedi (1738) and Linnaeus (1746), the original type-series of the nominal species Cobitis barbatula is not composed (at least not listed) of examined, described and/or illustrated specimen(s) by the author himself (category T1primary syntypes, of Dubois & Ohler, 1997a: 310), but based on publications of several earlier authors in the works referred to in the original description (category T2secondary syntypes) or even referred to a precedent reference made by those earlier listed authors (category T3tertiary syntypes). In the absence of any explicitly mentioned lists of type-specimens, a posteriori syntypes should be identified and herein are assigned to all previous bibliograph references cited by Artedi's (1738) and Linnaeus's (1746) accounts, referred by Linnaeus (1758) as the basis of the description of the nominal taxon [Art. 72.4.1], as listed in chronological order, as follows: [1] Figulus (1540), [2] ‘Cobitis barbatula’of Rondelet (1555: pilc. 2. p. 204.), [3] ‘Cobitis fluviatilis barbatula’ of Gesner (1563: pisc. 404), [4] ‘Cobitis barbatula’of Aldrovandi (1613: ichth. 618), [5] ‘Cobitis fluviatilis’of Schonevelde (1624: ichth. 31), [6] ‘Cobitis barbatula’of Jonstonus (1649: pisc t. 26. f. 22), [7] ‘Cobitis barbatula’of Charletonus & Charleton (1668: onom. 157), [8] ‘Cobitis fluviatilis’of barbatula Willugby (1686: ichth. 265) and [9] ‘Cobitis fluviatilis barbatula’of Ray (1713: pisc. 124). Because none of the syntypes are extant, and mostly no information is provided on lost specimens, they do not help to solve the nomenclature question. Noticeably, the drawings are not the syntypes but the specimens from which the drawings are made. Artedi's (1738) account is based on literature accounts by Figulus (1540), Rondelet (1555), Gesner (1563), Aldrovandi (1613), Schonevelde (1624), Willughby (1686), Ray (1713), Jonstonus (1649) and Charletonus and Charleton (1668). Linnaeus' (1746) account is based on Artedi (1738) and on the same accounts by Rondelet, Gesner, Aldrovandi, Jonstonus, Charletonus, Schonevelde, Willughby and Ray. Most of these sources, again, refer to material and older sources that we might have not investigated, which are all largely uninformative and give no exact geographic place or show a figure of a fish clearly stating its origin. Barbatula barbatula is based on a syntype series that consists of all the specimens above included by Linnaeus (whether directly or by bibliographic reference) (ICZN art. 72.4) and because there has been no holotype or lectotype designation, all these specimens are syntypes (art. 73.2). However, it is not possible to consistently associate the biological taxon of Cobitis barbatula of Linnaeus (1758) with a single well-identified taxon from the syntypes once they are composed of specimens belonging to distinct species-series taxa. All above-mentioned literature accounts are already enough to ensure that several species around Europe have been included in Linnaeus' original description of Cobitis barbatula. These currently include populations potentially from France =Barbatula sp. (Rondelet), Languedoc =Barbatula quignardi (Rondelet), Germany, Switzerland =Barbatula sp. (Gesner), northern Italy =B. pironae (Aldrovandi), England =Barbatula sp. (Willughby, Ray, Charletonus), probably Rhine drainage near Frankfurt =Barbatula sp. (Jonstonus), Schlesswig =Barbatula sp. (Schonevelde) and ‘Europae fluvii’=several species (rivers of Europe; Linnaeus, 1758). Undescribed species cited above are recognised based on the molecular results of this and previous studies (Barluenga & Meyer, 2005; Behrmann et al., 2017; Clavero et al., 2023; Denys et al., 2021; Sedivá et al., 2008; Knebelsberger et al., 2015; Norén et al., 2017; Zangl et al., 2020). Thus, the procedure to first designate one of the lost old specimens of unknown origin as lectotype (virtual lectotype–Dubois & Ohler, 1995,1997a;Dubois& Ohler, 1997b;Dubois,2011) and in a second step designate a neotype from a precise locality is appropriate given that the original syntypes are heterogeneous as herein recognised and non-extant. The taxonomic situation of B. barbatula is similar to that found in Gobio gobio (Kottelat & Persat, 2005), Cottus gobio (Freyhof et al., 2005), Coregonus oxyrinchus (Freyhof & Schöter, 2005) and Phoxinus phoxinus (Kottelat and Freyhof 2007). To clarify the nomenclature of B. barbatula, we follow the same approach of these previous studies, which is also reinforced by the recommendation of Dubois et al. (2014) for this kind of case. The virtual lectotype designation procedure as intermediate step is needed to deprive NRM 68 of its potential but questionable status as syntypes. The syntype series of Cobitis barbatula includes no references giving specific data of an explicitly mentioned specimen. Most of the above-mentioned authors from the syntype series mention the species description in vague words and it is usually not clear if the information is original or compiled, thus these cannot be used as a criteria for the selection of a lectotype. However, Rondelet (1555), Gesner (1563), Aldrovandi (1613), Willughby (1686) and Jonstonus (1649) show a figure of Cobitis barbatula in their work. Gesner (1563) reproduced and cited the figure from Rondelet, as well as a second figure likely to have been drawn for his book. Aldrovandi (1613) also copied the figure from Rondelet (1555), and Jonstonus and Jonston (1649) copied the figures from Rondelet (1555) and Gesner (1563). Willughby (1686) shows along the description of the nomina Cobitis fluviatilis barbatula, a figure citing the species form Cobitis barbatula fundulus of Baltneri (1666), which likely means that he copied Baltneri's figure. Therefore, Cobitis barbatula has secondary and tertiary syntypes in the original syntypic series, but non-extant, and it consists of at least three individuals that might have been originally drawn independently for the books by Rondelet (1555), Gesner (1563) and Willughby (1686). However, despite not being mentioned in the text or illustration, we cannot exclude that these authors copied their figure from another older source, and we also cannot exclude that some figures were composite, based on more different specimens. Gesner (1563), who based his description on the figure of Rondelet (1555), mentioned Cobitis barbatula under the common name Grundel/Bartgrundel, stating that it is a ‘common fish known from many people in our country (read here Switzerland)/occurs in several species’. Gesner (1563: pisc. 404) observed in the description that ‘some of them have barbels/and this is name-giving (from such their names)/others have in place of the same points thorns’, which lead us CALEGARI ET AL.5 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License to assume that the material he examined was not only composed of distinct Barbatula species, but might have also included specimens nowadays assigned to the genus Cobitis. Gesner mentioned that these fishes were particularly ‘common in the ‘Glatt’, but also caught in the Thöss, on the shore of lake Zurich, and near 'Arau' in the Aare, which are the largest individuals’. However, this geographical area is occupied by more than one species of Barbatula and also Cobitis bilineata. Even more complex, those areas represent contact zones between different species, where we cannot rule out the existence of hybrid individuals, therefore the information from these localities was not used as a criterion to guide the choice of a lectotype because it was unclear which species was meant. To refer unambiguously to the nominal species Cobitis barbatula, we choose here from the syntype series the specimen on which the drawing in Rondelet (1555) was based on. This is the first work to mention Cobitis barbatula and was the basis for the subsequent bibliographic references that mentioned that species. The specimen of Rondelet (1555) is here designated as lectotype of Cobitis barbatula.If NRM 68 were syntypes, they are now paralectotypes and they can no longer be used for a lectotype designation. The type locality of Cobitis barbatula is restricted to the locality of the lectotype. Rondelet (1555) gave no information on the locality of his specimen(s). As the typelocality has been restricted by the lectotype designation, the neotype should have originated from ‘as nearly as practicable from the type locality’(Article 75.3.6). Rondelet was born and lived in Montpellier (France), studied in Paris and Montpellier, and practiced as medical doctor in Maringues for a while before returning to live in Montpellier. He travelled extensively through France, to Amsterdam and through northern Italy, studying fishes, but his long residence in sight of the Mediterranean probably influenced him to study fishes from this region (Gudger, 1934). He corresponded with other naturalists, for example with Conrad Gesner, who worked mostly in Germany and Switzerland. It is not known where exactly he collected the lectotype of C. barbatula. We might speculate that the specimen came from France because it is a common species there and Rondelet might have found it virtually everywhere, even likely from the Mediterranean basin since Cuvier stated that Rondelet publication was the outstanding work on Mediterranean fishes until that of Risso in 1810 (Gudger, 1934). However, specimens might have even been sent to him. The collection made by Rondelet, if there was any, has not survived. The lectotype is thus not extant. To stabilise the nomenclatural status and the consequent use of the nomen B. barbatula, we need to designate an identifiable neotype of a known origin to clarify the identity of the nominal species Cobitis barbatula. We choose the population adjacent to Montpellier (France), where Rondelet spent most of his life. This population is known under the name B.quignardi, which has become now a junior synonym of B. barbatula. We designate here as neotype the specimen MNHN-IC-2024-1386, 46.2 mm SL, from the Lez River (France), which now becomes the type locality of C. barbatula. This individual belongs to what is presently called Barbatula barbatula (ICZN art. 75.3.5). The diagnosis and description are given below in detail (art. 75.3.2) and the unique catalogue number allows identification of the neotype (art. 75.3.3), which corresponds to what we know of the former types (art. 75.3.5) and comes from a locality ‘as nearly as practicable’from the original [Rondelet] type locality (art. 75.3.6) and is now deposited in the MNHN ichthyology collection (art. 75.3.7). Barbatula barbatula (Linnaeus) (Figure 2) Cobitis barbatula Linnaeus, 1758: 303 (type locality: France, Lez River; neotype: MNHN-IC-2024-1386, by present designation). Noemacheilus barbatulus quignardi B acescu-Mes¸ter [L.] 1967:359 (type locality: Le Lez River, near Montpellier, France; holotype: MGAB 77, paratype: MGAB 78, collected with holotype). Neotype. MNHN-IC-2024-1386, 46.2 mm SL, male, France, Lez River, Mediterranean Basin, Hérault Department, no coordinates, 1977, Quignard, J. P. Material examined. MNHN-IC-1977-0134, 2, 37.9–44.4 mm SL, topotypes, collected with neotype. MNHN-IC2010-1064, 6 of 7 alc. (1 adult measured, 39.8 mm SL) +1 mitogenome (voucher tag FFFtag4260, 41.7 mm SL, GenBank Accession Number MW288293), collected in the type locality of B.quignardi, France, Lez River at Prades-le-Lez, Dept. Hérault, 43420000 N, 351057.600 E, 24 Nov 2010, Denys, G. and Office National de l'Eau et des Milieux Aquatiques (ONEMA). MNHN-IC-2021-0358, 1, France, Dept. Hérault, 2021, Denys, G. MNCN-ICTIO 17741–17742, 2, France, Isle, Sorgue River, tributary of the Rhône River, 441305900 N, 59026.5000 E (approx. coordinates), 30 Apr 1968, unknown collector. Diagnosis.Barbatula barbatula is a member of a monophyletic group of species, herein designated as Western Europe clade (including B. hispanica,B. leoparda,B. fluvicola n. sp. and B.ommata n. sp.), which is diagnosed from all its valid congeners by having a longer dorsal head length 23.2%–25.8% SL (vs. 19%–21.8% in B. hispanica, 20%–21.5% in B. leoparda, 18.4%–21.7% in B. fluvicola n. sp. and 19.3%–21.4% SL in B.ommata n. sp.), longer lateral head length 25.9%–29.6% SL (vs. 22%–24.9%, 22.5%–25%, 21.8%–24.5% and 22.4%–26.2% SL, respectively), longer predorsal length 55.8%–60.4% SL (vs. 49.8%–53.3%, 53.5%–54.4%, 50.5%–55.8% and 50.4%–56.8% SL, respectively), longer prepectoral length 26.2%–28.7% SL (vs. 20.8%–25%, 22.2%–24.7%, 20.9%–25.1% and 21.3%–25.8% SL, respectively), longer dorsal fin 24.1%–25.8% SL (vs. 21.2%–23.8%, 20.4%–23.4%, 18.4%–24.2% and 18.4%–23.3% SL, respectively), shorter caudal peduncle 11.7%–12.7% SL (vs. 13.7%–17.4%, 12%– 14.4%, 13.9%–17.3% and 12.7%–16.2% SL, respectively), and protuberant slope present from preceding area of anterior nares (vs. slope preceding anterior nares absent or weakly developed). FIGURE 2 Lateral view of the neotype of Barbatula barbatula, MNHN-IC-2024-1386 [Correction added on 8 August 2025, after first online publication. The specimen collection number in the preceding text has been corrected in this version.], 46.2 mm SL, male, Lez River, France. 6CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License It is distinguished from B. leoparda,B. fluvicola n. sp. and B.ommata n. sp. by having longer dorsal-fin base 14.5–18.6% SL (vs. 12.8%– 14.4% in B. leoparda, 11.2%–13.6% SL in B. fluvicola n. sp. and 11.1%– 14.3% in B.ommata n. sp.) and from B. hispanica,B. fluvicola n. sp. and B. ommata n. sp. by having deeper head 51.7%–57.3% HL (vs. 40.2%– 49.3%, 46.1%–51.7% and 38.4%–46% HL, respectively). Barbatula barbatula is further distinguished from B. fluvicola n. sp. and B. ommata n. sp. by having the adpressed anal-fin tip slightly anterior to end of caudal peduncle (vs. adpressed anal-fin tip reaching about middle of caudal peduncle) and larger suborbital depth 33.1%–36.3% HL (vs. 26.5%– 32.4% and 23.7%–31.2% HL). It further differs from B.leoparda by having 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); and from B. ommata n. sp. by having a longer first dorsal-fin ray 20%–24.3%SL (vs. 13.5%–19.8% SL) and a longer snout 50.8%–52.6% HL (vs. 37.2%–46.1% HL). Redescription. Morphometric data are shown in Table 1. General appearance is shown in Figure 2. Complementary description details TABLE 1 Morphometric data for Barbatula barbatula,n=3 specimens, range includes neotype (Neo). Neo Min Max Mean SD Standard length (mm) 46.2 39.8 46.2 43.5 - Percentage of standard length Dorsal head length 25.8 23.2 25.8 24.3 1.3 Lateral head length 29.6 25.9 29.6 27.8 1.9 Predorsal length 60.4 55.8 60.4 57.5 2.5 Postdorsal length 32.3 32.3 32.4 32.3 0.1 Prepectoral length 28.7 26.2 28.7 27.5 1.3 Prepelvic length 59.5 55.9 59.6 58.3 2.1 Preanal length 83.0 81.0 83.0 82.1 1.0 Pectoral-fin origin to pelvic-fin origin 33.8 30.9 33.8 32.4 1.5 Pelvic-fin origin to anal-fin origin 26.0 23.3 26.0 24.8 1.4 First dorsal-fin ray length 20.0 20.0 24.3 22.4 2.2 Dorsal fin length 25.4 24.1 25.8 25.1 0.9 Dorsal-fin base length 18.6 14.5 18.6 16.0 2.2 Anal-fin base length 11.8 8.3 11.8 9.7 1.8 First pectoral-fin ray length 22.3 17.7 22.3 20.0 3.3 Pectoral fin length 26.7 20.4 26.7 23.5 4.4 First pelvic-fin ray length 14.5 13.1 17.6 15.1 2.3 Pelvic fin length 19.3 17.2 19.3 18.3 1.1 Caudal-peduncle length 12.5 11.7 12.7 12.3 0.5 Caudal-peduncle depth 14.9 9.8 14.9 12.3 2.5 Caudal-peduncle width 2.6 1.7 2.6 2.2 0.4 Body depth at pectoral-fin origin 16.4 14.3 16.6 15.8 1.3 Body width at dorsal-fin origin 12.7 11.8 13.0 12.5 0.6 Percentage of head length Head depth 54.3 51.7 57.3 54.4 2.8 Snout length 52.2 50.8 52.6 51.9 1.0 Postorbital length 50.9 47.0 54.2 50.7 3.6 Orbital diameter 23.0 19.4 24.7 22.4 2.8 Suborbital depth 36.3 33.1 36.3 34.2 1.8 Interorbital distance 31.9 30.1 34.2 32.0 2.1 Anterior internarial distance 16.0 12.8 16.8 15.2 2.1 Posterior internarial distance 27.1 23.0 27.1 25.5 2.2 Upper–lower lips distance 29.7 23.0 29.7 26.9 3.5 Lower lip length 21.2 16.6 21.2 18.4 2.4 Mental lobe length 9.4 9.3 9.9 9.5 0.3 Mouth width 36.4 31.4 36.4 34.6 2.8 Abbreviations: Max, maximum; Min, minimum; SD, standard deviation. CALEGARI ET AL.7 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License are shown in the original description of Barbatula b. quignardi (B acescu-Mes¸ter, 1967). Body short and relatively thickened, roughly cylindrical in cross-section at trunk, gradually compressed towards caudal peduncle. Dorsal profile of head straight rising from snout tip to posterior border of supraoccipital; conspicuous hump demarking posterior border of supraoccipital. Ventral profile of trunk slightly convex. Body depth slightly decreasing from end of dorsal fin towards caudal-fin base. Greatest body depth at predorsal area. Greatest body width at opercle or preceding pectoral-fin origin. Head relatively deep, cylindrical in dorsal view. Dorsal head profile with protuberant slope present from preceding area of anterior nares, larger than congeners. Anterior border of snout rounded in dorsal view, tapering towards its tip; anterior portion truncated. Eyes laterodorsally positioned; positioned dorsal half on head. Orbital rim free. Nostrils small. Anterior nostril surrounded by elongate fleshy flap-like tube integument; fleshy flap elongated. Posterior nostril without fleshy flap and located close to anterior border of eye. Anterior and posterior nostril closely set, positioned almost continuously and connected by thin skin ridge. Gill openings united to isthmus laterally approximately at line of inner portion of pectoral-fin base origin. Mouth ventral, arched in a semicircular shape. Upper lip thin; vertical median incision (notch) not evident. Contralateral lower lips separated at midline by a deep notch. Mesial portion of lower lip with well-developed mental lobe forming a fleshy fold, but relatively short; conical protrusion of mental lobe absent. Lateral lobe or projection of lateral portion of lower lips absent. Maxillary barbel longer than premaxillary barbel pairs, surpassing vertical of middle of eye. Two pairs of premaxillary barbel. Mesial premaxillary barbel tip usually not reaching end of mental lobe of lower lip. Lateral premaxillary barbel positioned laterally on snout, longest than mesial barbel; its tip reaching middle of posterior naris. Pectoral-fin rays i,10*(1), i,11*(1) or 12*(1); distal margin acute, mesial border somewhat convex. Epithelial tubercles present in pectoral-fin pterygiophores and increase in size towards pectoral-fin tip. Pelvic-fin distal border ellipsoid; pectoral origin situated midbody length, at vertical of second branched dorsal-fin ray. Pelvic-fin rays i,6 (1) or I,7*(2). Axillary pelvic lobe present and conspicuous. Dorsal fin long compared to congeners; its origin located slightly posteriorly midbody; fin tip truncated. Dorsal-fin rays i,7½*(3). Anal fin i,5½*(3) rays. Anal fin with distal margin truncated. Anal-fin origin located at third half of body length, posterior to end of ultimate branched dorsal-fin ray. Caudal fin emarginate; upper and lower lobes similar in length. Caudal-fin rays i,8 +8,i* (3). Caudal-fin peduncle short and deep, approximately same depth as the remain part of body and head. Procurrent rays pronounced and embedded in the skin forming a dorsal and ventral dermal keel extended two thirds or more of caudal peduncle. Body covered by embedded, tiny, roundish scales, not overlapping each other. Lateral line incomplete and continuous, reaching anterior portion of anal-fin base. Coloration (in alcohol). Background colour yellowish paleto light brown. Head surface mottled, sparsely set and fainted towards cheek and lateral part of snout. Dorsolateral portion of body with conspicuous, irregular set five dark-brown blotches (Figure 2). Flank with conspicuous, large, dark-brown, irregular rounded blotches, usually separated from each other. Pigmentation fading below lateral line. Ventral surface of body and head mostly without pigmentation, except for caudal-fin peduncle with few sparsely blotches. Dorsal surface of pectoral-fin base and dorsal-fin base densely dark-brown pigmented. Pigmentation on barbels and lips restricted to few spots and small blotches. Pectoral-fin pterygiophores with elongate dark-brown blotches forming two to three elliptical lines. Dorsal fin with pigmentation concentrated in its base; rays with small, somewhat rectangular dark-brown blotches on all rays, usually arranged in rows. Pelvic-fin rays hyaline or with very faint and scattered pigmentation in outermost rays. Anal fin hyaline. Caudal-fin base with dark-brown, slightly m-shaped bar in middle and comma-shaped blotch on upper caudalfin base. Caudal fin covered by small, dark-brown, elongate blotches forming vertical series or are randomly distributed all over caudal fin. Sexual dimorphism.Barbatula barbatula shows pectoral fin elongated, acute in adult males (vs. elliptical to rounded in females and juveniles). In males, second branched ray longest than remaining (vs. third branched ray longest than remaining in females). Welldeveloped epidermical tubercles in the pectoral-fin rays of adult males (absent in females and not evident in juvenile males). Tubercles present in the principal unbranched ray and 1–6 innermost branched rays, rare in eighth ray (Figure 3in B acescu-Mes¸ter, 1967). Urogenital papilla of males small and conical, and urogenital opening in females rounded; in both sexes, smaller in size when compared to its congeners; more difficult to distinguish the sexes. Distribution.Barbatula barbatula is currently confirmed to occur in the Lez River in France (see Nomenclature section) and the Sorgue River (based on morphological identification; Figure 3). The presence of the species, confirmed by coI barcode in the lowermost part of the Rhône drainage, is likely to indicate its presence in other surrounding areas. The range of the species may therefore be wider than currently known (Figure 3). Remarks. Populations from the Doubs and Allaine rivers in the upper portion of Rhône in Switzerland were recovered nested in the B.barbatula clade based on coI gene, but exhibited significant morphological differences compared to B.barbatula populations from the Lez and Sorgue rivers. Additionally, part of the populations from the rivers Le Bied, Broye and Areuse Travor, disconnected streams from the remaining Aare catchment in Switzerland, were also recovered in the coI tree nested within B.barbatula population from the Lez river, although we were unable to check their morphology. Additional material is required to determine whether they are conspecific and to precisely define the geographical range of B.barbatula. Barbatula fluvicola, new species (Figures 4and 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, 8CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 3 Legend on next page. CALEGARI ET AL.9 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 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 FIGURE 10 Polymorphic colour pattern in different ontogenetic stages of preserved specimens of Barbatula fluvicola: Lyssbach River, Rhine drainage (a) NMBE 1105475, 72.3 mm SL, (b) NMBE 1105479, 69.45 mm SL, (c) NMBE 1105485, 56.38 mm SL; and Limpach River, Rhine drainage (d) NMBE 1105345, 38.4 mm SL. Scale bar =1 cm. 16 CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 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 nontype 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), NordVoralpine 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C; 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 FIGURE 11 Dorsal, left lateral and ventral view of Barbatula ommata, holotype, NMBE 1105555, 74.1 mm SL, Lake Neuchatel, Aare drainage, Rhine River system, Switzerland. FIGURE 12 Live coloration of B.ommata, (a) NMBE 1105555 (holotype), (b) NMBE 1105293, type locality, Lake Neuchatel, and (c) NMBE 110523, Lake Lucerne. CALEGARI ET AL.17 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License conducted for better understanding of its abundance and population stability. Barbatula ommata, new species (Figures 11 and 12) urn:lsid:zoobank.org: act:4A1BCF03-B054-41B6-8020-F34B01B13ADE Barbatula sp. ‘Lineage I’–Alexander & Seehausen 2021:110–111 [listed]. Holotype. NMBE 1105555, 74.1 mm SL, female, Corcelles-prèsConcise beach, Lake Neuchatel, Aare catchment, Rhine drainage, Canton of Vaud, Switzerland, 465003300 N, 64204200 E, 20 Jul 2023, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. Paratype. All from Switzerland: Rhine drainage: Aare catchment: Canton of Vaud: NMBE 1105553–1105554, 2, 69.1–73.7 mm SL, collected with the holotype. —NMBE 1105282–1105290, 9, 53.1– 70.9 mm SL, Lake Neuchatel, 465104700 N, 65103800 E, 19 Jul 2023, Calegari, B. B., Wegscheider, B., Waldock, C., Josi, D. —NMBE 1105291–1105293, 3, 56.9–70.7 mm SL, Lake Neuchatel, Corcellesprès-Concise beach, 465004800 N, 64205900 E, 20 Jul 2023, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. Canton of Neuchâtel: NMBE 1105309–1105311, NMBE 1105313, NMBE 1105315– 1105326, NMBE 1105328, 17, 46.8–57.6 mm SL (HRXCT of NMBE 1105313), NMB 6482–6483, 2, 52.7–54.5 mm SL, ZFMK-ICH 135312, 1 C&S, 54.6 mm SL, Lake Neuchatel, littoral zone, 367 m alt., 47008.500 N, 7004000 E, 17 Jul 2022, Calegari, B. B., Wegscheider, B., Josi, D. —MHNN 89-2491 and MHNN 89-2492, 2, 64.1–67.2 mm SL, Lake Neuchatel, littoral zone at camping Paradis-Plage, 465800.400 N, 652025.500 E, 16 Oct 2024, Calegari, B. B., Sudasinghe, H. Canton of Fribourg: NMBE 1105261–1105264, NMBE 1105266– 1105269, NMBE 1105272–1,105,278, NMBE 1105280, 48.7– 66.2 mm SL, ZFMK-ICH 135309–135,311, 3, 62.9–65.6 mm SL, ZSM 49671, 2, 53.7–56.4 mm SL, NMW 101169, 2, 52.4–65.9 mm SL, Lake Neuchatel at Estavayer harbour, 464903100 N, 64006000 E, 28 Jun 2023, Calegari, B. B., Wegscheider, B., Josi, D., Waldock, C. Canton of St. Gallen: NMBE 1105245–1105249, 5, 49.1–75.7 mm SL, Lake Walen, near to Quinten, 47705700 N, 91103200 E, 28 Jun 2023, Calegari, B. B., Wegscheider, B., Josi, D., Reichlin, P. —NMBE 1105237– 1105238, NMBE 1105241–1,105,242, NMBE 1105244, 6, 51.3– 82.3 mm SL, ZFMK-ICH 135313–135314, 2, 53.6–71.5 mm SL, Lake Walen, near to Quinten, 47705200 N, 9120200 E, 28 Jun 2023, Calegari, B. B., Wegscheider, B., Josi, D., Reichlin, P. —NMBE 1105250– 1105258, 8 +1 C&S, 56.7–71.7 mm SL, Lake Walen, near to Mols, 476051.400 N, 916055.800 E, 28 Jun 2023, Calegari, B. B., Wegscheider, B., Josi, D., Reichlin, P. Canton of Lucerne: NMBE 1105219– 1105221, NMBE 1105224–1105226, NMBE 1105228–1105229, NMBE 1105231–1105235, 12 +1 C&S, 50.6–71.1 mm SL, MNHN 2024–1385, 2, 55.0–63.8 mm SL, MNCN-ICTIO 298.404 and MNCNICTIO 298.405, 2, 56.5–68.2 mm SL, Lake Lucerne at Vitznau, 430 m alt., 47001400 N, 9290400 E, 27 Jun 2023, Calegari, B. B., Wegscheider, B., Josi, D., Reichlin, P. Canton of Nidwalden: NMBE 1105236, 1, 58.5 mm SL, Lake Lucerne, Beckenried, 440 m alt., 465801800 N, 82605700 E, 27 Jun 2023, Calegari, B. B., Wegscheider, B., Josi, D., Reichlin, P. Genseq-2 coI. NMBE 1105553 (tissue tag 241420); Genbank Acession Number PQ488228. Genseq-2 coI. NMBE 1105282 (tissue tag 241424); Genbank Acession Number PQ488229. Genseq-2 coI. NMBE 1105291 (tissue tag 241441); Genbank Acession Number PQ488230. Genseq-2 coI. NMBE 1105310 (tissue tag 239503); Genbank Acession Number PQ488120. Genseq-2 coI. NMBE 1105245 (tissue tag 241280); Genbank Acession Number PQ488223. Genseq-2 coI. NMBE 1105248 (tissue tag 241283); Genbank Acession Number PQ488224. Genseq-2 coI. NMBE 1105250 (tissue tag 241286); Genbank Acession Number PQ488225. Genseq-2 coI. NMBE 1105261 (tissue tag 241392); Genbank Acession Number PQ488227. Genseq-2 coI. NMW 101169 (tissue tag 241410); Genbank Acession Number PQ488226. Genseq-2 coI. NMBE 1105237 (tissue tag 241240); Genbank Acession Number PQ488221. Genseq-2 coI. NMBE 1105220 (tissue tag 241212); Genbank Acession Number PQ488217. Genseq-2 coI. MNCN-ICTIO 298.404 (tissue tag 241214); Genbank Acession Number PQ488218. Genseq-2 coI. NMBE 1105226 (tissue tag 241218); Genbank Acession Number PQ488219. Genseq-2 coI. NMBE 1105236 (tissue tag 241228); Genbank Acession Number PQ488220. Additional material (non-types).All from Switzerland. Canton of Bern: NMBE 1120429 and NMBE 1120489, 2, Lake Biel, 57.4– 58.7 mm SL, 470404800 N, 711046.700 E, 20 Sep 2017, Décourcière, H. —NMBE 1120476 and NMBE 1120485, 2, 53.5–59 mm SL, Lake Biel, 4703039.500 N, 710029.300 E, 20 Sep 2017, Décourcière, H. Canton of Fribourg: NMBE 1061957, NMBE 1061974, NMBE 1061976–1061978, NMBE 1061993–1061995, NMBE 1062005, 7 +1 C&S, 53.2–76.3 mm SL, Lake Neuchatel, 4652014.900 N, 652016.500 E, 3 Oct 2011, unknown collector. —NMBE 1061958, 1, 64.2 mm SL, Lake Neuchatel, at Sous la Corbière, 4651056.900 N, 651050.300 E, 3 Oct 2011, Project Lac. —NMBE 1074762, 1, 52.3 mm SL, Lake Neuchatel, at Sous la Corbière, 465205.900 N, 651011.100 E, 7 Oct 2011, unknown collector. Canton of Neuchâtel: NMBE 1074763, 1, 53.6 mm SL, Lake Neuchatel, at La Grande Béroche, 465506.900 N, 648055.600 E, 7 Oct 2011, Project Lac. Canton of Lucerne: NMBE 1069200–1069202, 3, 54.0–60.3 mm SL, Lake Lucerne at Vitznau, 430 m alt., 47001600 N, 8290100 E, 20 Aug 2014, Vonlanthen, P. —NMBE 1066769–1066772, 4, 36.3–40.3 mm SL, Lake Zug, near to Immensee, 470603000 N, 82806000 E, 22 Aug 2013, Tourreau, G. —NMBE 1080978, 1, 59.9 mm SL, Steinibach, 471014.500 N, 817059.900 E, 9 Sep 2013, Vonlanthen, P. —NMBE 1080958–1080971, 14, 58.2–76.0 mm SL, Steinibach, Ringstrasse, 470054.500 N, 818035.500 E, 9 Sep 2013, Vonlanthen, P. Canton of St. Gallen: NMBE 1074613, 1, 69.3 mm SL, Lake Walen, near to Sargans, 470704900 N, 912016.300 E, 23 Oct 2012, Brodersee, J. —NMBE 1111831–111833, NMBE 1109966, 4, 32.1–76.1 mm SL, Rufibach River, Limmat drainage, northwest of the airport Schänis between Maseltrangen and Ussbühl, 413 m alt., 4710043.700 N, 91044.800 E, 13 Sep 2022, Calegari, B. B., Wegscheider, B.; Josi, D., Sudasinghe, H. Canton of Schwyz: NMBE 1105516–1105530, 15, 54.9–88.8 mm SL, Canton of Schwyz, Hoggibach River, at side channel of Hintergraben/Linthkanal, Aare River, Reichenburg, 406 m alt., 4710032.700 N, 859021.900 E, 14 Oct 2022, Calegari, B. B., Wegscheider, B., Josi, D., 18 CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Waldock, C., Schmid, A., Moreau, S. Canton of Vaud: NMBE 1061953, NMBE 1061974–1061975, NMBE 1061992, NMBE 1062001, 4 +1 C&S, 52.2–57.6 mm SL, Lake Neuchatel, at Corcelles-près-Concise beach, 4650016.900 N, 642027.900 E, 3 Oct 2011, Project Lac. —NMBE 1061963 and NMBE 1061989, 2, 54.4–55.3 mm SL, Lake Neuchatel, at Corcelles-près-Concise beach, 465008.700 N, 642012.300 E, 3 Oct 2011, Project Lac. —NMBE 1062004, 1, 59.5 mm SL, Lake Neuchatel, near to La Poissine, 4649035.100 N, 64109.100 E, 3 Oct 2011, Project Lac. Canton of Zürich: NMBE 1105329–1105338, 9 +1 C&S, 36.9– 51.6 mm SL, Lake Zurich at Linthkanal, 394 m alt., 471306.200 N, 856028.900 E, 11 Jul 2022, Calegari, B. B., Wegscheider, B., Josi, D. — NMBE 1071771, NMBE 1071774, NMBE 1071787, NMBE 1071789, NMBE 1071793–1071794, NMBE 1071797, 7, 36.6–49.8 mm SL, Lake Zurich, Richterswill, 471301000 N, 841033.300 E, 1 Oct 2014, Vonlanthen, P. Diagnosis.Barbatula ommata is a member of a monophyletic group of species, herein designated as Western Europe clade (including B.barbatula,B. hispanica,B. leoparda and B. fluvicola), which is diagnosed from all clade congeners, except by B.hispanica, by having a shallower head 38.4%–46% HL (vs. 46.1–51.7% in B. fluvicola,51.7–57.3% in B.barbatula and 51.5–58.3% HL in B.leoparda). The new species differs from B.hispanica and B.fluvicola by having a larger eye 21.1%–28.3% HL (vs. 16.8%–22.5% and 16.1%– 19.8% HL). It is distinguished from B.leoparda and B.hispanica by having a shallower caudal peduncle 7.7%–10.9% SL (vs. 12.1%– 13.6% and 12.1%–13.9% SL). It is further distinguished from B.leoparda and B.barbatula by having a smaller suborbital depth 23.7%– 31.2% HL (vs. 32.2%–36.3% and 33.1%–36.3% HL). It further differs from B.leoparda by having a shorter lower lip 12.7%–17.1% HL (vs. 17.4%–21.2% HL), and ventral surface of head and area between pectoral to pelvic-fin origins without pigmentation (vs. ventral surfaces with brown to dark irregular blotches). B. ommata is further distinguished from B.barbatula by having a shorter dorsal head length 19.3%–21.4% SL (vs. 23.2%–25.8% SL), a shorter first dorsal-fin ray 13.5%–19.8% SL (vs. 20%–24.3% SL), a shorter dorsal-fin base 11.1%–14.3% SL (vs. 14.5%–18.6% SL), a shorter dorsal fin length 18.4%–23.3% SL (vs. 24.1%–25.8% SL), a shorter prepectoral length 21.3%–25.8% SL (vs. 26.2%–28.7% SL), a longer caudal peduncle 12.7%–16.2% SL (vs. 11.7%–12.7% SL), ashortersnoutlength37.2%–46.1% HL (vs. 50.8%–52.6% HL), a weekly developed slope present between anterior nares (vs. protuberant slope 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). Barbatula ommata differs from B.fluvicola, which have widely overlapping distribution ranges, by having ventral border of dentary straight (vs. ventral border of dentary curved; Figure 8), shorter coracoid, halflength of cleithrum (vs. coracoid long, occupying at least two thirds of cleithrum length; Figure 7), larger swimbladder bony capsule, occupying slightly more than half of head depth (Figure 13) (vs. smaller swimbladder bony capsule, not reaching half of head depth), and posterior border of swimbladder bony capsule rounded in ventral view (Figure 13) (vs. posterior border of swimbladder bony capsule straight in ventral view). Description. Morphometric data and counts are provided in Table 2. Body robust and elongate, cylindrical in cross-section at trunk. Dorsal profile of trunk approximately straight descending from dorsal-fin origin to middle of caudal-fin peduncle; ascending from that point to caudal-fin origin. Ventral profile of trunk straight, except from anal fin descending towards caudal-fin end. Dorsal head profile slightly concave from snout tip to posterior border of supraoccipital. Trunk depth similar along its length slightly decreasing posteriorly dorsal-fin base towards middle of caudal-fin peduncle. Greatest body depth slightly anterior to dorsal-fin origin. Body width decreasing and FIGURE 13 HRXCT model of skull and anterior body of Barbatula ommata, NMBE 1105313, paratype, 50.8 mm SL. (a) Dorsal view. (b) Lateral view of left side. aar, anguloarticular; ant, antorbital; apa, autopalatine; ba, basioccipital; br, branchiostegal rays; bsb, bony capsule of swimbladder; cl, cleithrum; co, coracoid; cv, complex vertebrae; den, dentary; ect, ectopterygoid; en, endopterygoid; epo, epioccipital; exo, exoccipital; fr, frontal; hyo, hyomandibula; io, interopercle; ki, kinethmoid; let, lateral ethmoid; max, maxilla; met, metapterygoid; nc, neural complex; nv-4, neural arch of vertebrae four; op, opercle; orb, orbitosphenoid; pa, parietal; peth-II, preethmoid two; pfr, pectoral-fin rays; pmx, premaxilla; pop, preopercle; ps, parasphenoid; pro, prootic; pto, pterotic; pts, pterosphenoid; pv, prevomer; qua, quadrate; rad, pectoral-fin radial; rar, retroarticular; ptt, posttemporal; sca, scapula; se, supraethmoid-ethmoid; scl, supracleithrum; soc, supraoccipital; spo, sphenotic; sop, subopercle; sym, sympletic. Scale bar: 2 mm. CALEGARI ET AL.19 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License gradually compressed from pectoral-fin origin towards caudal fin. Greatest body width at dorsal-fin origin. Head cylindrical in dorsal view, longer than wide. Eyes laterodorsally positioned, located at middle of head length. Ocular capsule rounded to slightly ellipsoid. Nostrils small; anterior naris opening slightly smaller than posterior one. Anterior nostril with an elongate fleshy flap-like tube oriented laterodorsally. Posterior naris in oval shape with anterior border slightly pointed. Anterior and posterior nostril close to each other and connected by thin skin ridge. Ventral border of gill opening attached to body at line of pectoral-fin base. Mouth ventral, arched in a semicircular shape. Mouth wide, almost equal to head width. Gape twice times longer than deep. Upper lip somewhat slim, leaving exposed middle portion of premaxilla area. Median incision usually deep, occupying approximately entire lip depth; poorly developed in some individuals. Lower lip separately at midline by a deep notch. Mesial portion of lower lip developed in a fleshy fold in inverted U-shape forming a mental lobe. Mental lobes usually separate each other at mid-line along entire length; without protrusion. Anterior border of lower lips in majority of specimens not covering anterior border of dentary, leaving this portion visible. Lateral portion of lower lip positioned inclined in 45 angle to vertical midline, not bearing evident furrows; posterior tip without projection. Lower and upper lips rugous covered with small rounded papillae in its entire length. Barbels tapering gradually towards tips. Two pairs of premaxillary barbel of similar width. Tip of mesial premaxillary barbel reaching or almost reaching to base of maxillary barbel. Lateral premaxillary barbel tip surpassing middle of posterior naris usually reaching anterior border of eye. Maxillary barbel slightly surpassing vertical of middle of eye. Pectoral-fin rays i,11* (31). Innermost soft ray unbranched and tiny, usually visible only in C&S specimens. Distal margin of pectoral fin truncated or convex. Principal unbranched pectoral-fin ray slightly longer than two-thirds of longest pectoral-fin ray. Pelvic-fin rays i,7* (31). Pelvic fin ellipsoid, its origin at vertical through second branched dorsal-fin ray. Pelvic fin with distal margin convex, its tip slightly anterior to vertical of end of adpressed dorsal fin. Axillary pelvic lobe present, conspicuous attached to body, in oblate ellipsoid shape. Dorsal-fin origin located slightly after middle of body; distal margin slightly convex to truncated. Dorsal fin with 3 small (6 C&S), unbranched rays, and i,7½* (30) or i,8½ (1). Dorsal fin with 9 (4 C&S) or 10 (2 C&S) pterygiophores. First dorsal-fin pterygiophore composed by what seems a supraneural and first proximal radials fused each other in a single complex structure supporting 3 shorter simple rays +a principal longest unbranched ray. Remaining 8 or 9 pterygiophores with posterior laminar bony keel, supporting their respectively branched ray, except last pterygiophore lacking any correspondent ray, and smaller in size. Ultimate 1½ dorsal-fin rays supported by the penultimate pterygiophore. Principal dorsal-fin unbranched ray shorter than the subsequent outermost three branched rays, reaching two-thirds of its length. First dorsal-fin pterygiophore inserted anterior to neural spine of 13–14th vertebrae (6 C&S). Distal margin of anal fin slightly concave due to longer mid-rays. Anal fin with 2–3 shorter simple rays (6 C&S) plus i,5½* (25) rays. Two to 3 simple rays +first unbranched principal ray supported by composed structure of at least 3 proximal radials fused in a single element; remaining branched rays supported by 5 pterygiophores; ultimate pterygiophore supporting 1½ rays. Anal-fin origin located posterior to vertical of dorsal-fin end. First pterygiophore inserted anteriorly to hemal spine of 25th or 26th vertebra (6 C&S). Caudal fin weakly emarginate (rarely truncated). Caudal-fin rays i,7 +6,i (1), i,7 +7,i (2), i,8 +7,i * (23), or i,8 +8,i (5 C&S). Procurrent rays in dorsal lobe 7, and ventral lobe with 6 rays. Procurrent rays pronounced and embedded in skin forming a dorsal and ventral adipose keels extended on up to half of caudal-peduncle length. Ventral adipose keel slightly elevated, shorter than dorsal keel. Ventral margin of caudal fin sometimes somewhat convex, while dorsal margin approximately straight in most of specimens; rarely straight in both margins. Epural reaching half-length of caudal skeleton. Upper hypural plate as a single element with hypurals 3, 4 and 5 articulated, but not fused, separate each other along entire length. Lower hypural plate with co-ossified parhypural, and hypurals 1 and 2 separated each other, forming the compound caudal centrum. Total vertebrae, 39 (3 C&S) or 40 (3 C&S); 13 pairs of ribs (6 C&S) gradually decreasing length towards caudal fin. Body covered by embedded tiny, rounded, not overlapping scales, separated by a distance larger than scale diameter. Scales irregularly set along trunk. Scales present on flank from anterior vertical of dorsal-fin origin to caudal-fin base. Ventral portion of body with scales present only between end of pelvic-fin girdle to anal-fin origin. Scales completely absent on predorsal region and head. No evident developed dermal tubercles in any mature or juvenile individuals, even at reproductive season. Lateral line incomplete, ending before caudal-fin base approximately at vertical of end of anal fin. Lateral line quite variable with 32–55 (6 C&S) canals; first three to four canals well ossified. Lateral line interrupted usually at vertical through dorsal-fin origin continuing approximately at vertical of end of anal-fin base. Lateral line canals drastically decreasing size after first half of trunk and more spaced each other towards caudal fin. Cephalic lateral sensorial system with canals only partially ossified, between pores, sometimes weakly ossified. Infraorbital canal 11– 12 pores (two pores +posterior-half pore of antorbital segment, and anterior-half pore +nine pores +posterior-half pore of suborbital segment). Infraorbital canal composed by the antorbital branch bearing two pores (anterior and dorsomedial) and posteriorly half-pore, close but separately from anterior-half pore of adjacent suborbital branch, followed by nine to 10 pores and ending in posterior-half pore. Posterior-half pore of suborbital segment connected to postorbital canal with anterior-half pore, plus three pores and ending in posterior-half pore connected to half pore of both supratemporal and lateral line canal. Supratemporal canal with two and half pores. Nasal canal with three pores (anterior, mid-lateral, and posterior) usually unossified (rarely weakly ossified). Frontal canal bordering orbital border, but truncated at mid-portion splitting in two branches, each one bearing three pores (anterior, lateral and posterior). Preoperculomandibular canal with eight to nine pores with anterior part located ventrally to articular and posteriorly to dentary. Trunk canal short with 20 CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License two pores limited by pectoral-fin girdle. Nasal canal present with three pores (Figure 1). Colouration (in alcohol). Overall background colouration pale yellow. Body trunk covered by light to medium brown pigmentation forming irregular roundish blotches, randomly distributed (Figure 11). Pigmentation more concentrated on dorsal surface of body, with three to four brown blotches on predorsal region, and three blotches on postdorsal region, less evident in most individuals. Blotches mostly separately from each other, but coalescent. Lateral line portion usually bearing a faint brown stripe formed by coalescent blotches of pigmentation, sometimes more conspicuous and continuous at middle to end of trunk. Pigmentation fading abruptly in ventral portion of head and body, between pectoral-and-pelvic fins origins, and abdominal region. Head with more uniform dark-brown pigmentation along dorsal profile by concentration of pigmentation. Suborbital area and distal margin of opercle pale yellow to whitish with small, sparse pigmentation. Dark-brown, narrow stripe present on prenasal area, extending from snout tip to anterior portion of eye, but interrupted by nasal opening. Specimens varying in pigmentation over axillary pelvic-fin keel and at dorsal surface of pectoral-fin base, from barely present to conspicuous darker brown colour. Pigmentation absent on maxillary barbel, but present and faded in premaxillary barbels, usually restricted to base of barbel, rarely reaching its half length: mesial premaxillary barbel more pigmented than lateral pair. Upper and lower lips deprived of any pigmentation, except by the anterior area preceding upper lips. Pectoral fin with elongate dark brown blotches over rays reaching almost entire length, except by posterior margin. Outermost six pectoral-fin rays with larger blotches, more concentrate along anterior half of fin length; fainting towards mesial portion. Concentrated dark-brown pigmentation present on dorsal-fin base and at distal portion of fin; fading at middle portion of rays. Pigmentation on dorsal-fin rays forming or not rectangular dark bars over all rays, distributed irregularly; more conspicuous on first unbranched ray. Pelvic fin with no pigmentation or rarely showing few faint pigments concentrated only on distal portion of fin. Pigmentation on anal fin mostly absent, except by distal portion with faded small pigmentation. All fins lack pigmentation on ventral surface. Caudal-fin rays covered by small irregular pigmentation over entire length, except on distal margin. Elongated dark-brown to black blotch on caudal-fin base. Colouration in life. Similar to that observed in preserved specimens but with ground colour of body more pale yellow and mid-portion of lateral body with gold iridescent coloration in some individuals. Distribution.Barbatula ommata is known only from the lakes Neuchatel (Figure 6b), Lucerne, Walen, Zurich, Zug, Biel and Murten, all systems of the Aare drainage in Switzerland (Figure 3). The species has been further historically reported from lakes Thun and Brienz (Figure 3), but its continued occurrence in these systems remains uncertain due to the lack of recent records, highlighting the urgent need for further surveys to confirm its persistence and presence in these lakes. Remarks. There are four records of Barbatula in the InfoFauna database (www.infofauna.ch), dated 1988, 1998, 2001 and 2011, from two sites in Lake Thun and one record (1998) from Lake Brienz. These historical records lack accompanying photographs, voucher specimens or precise geographic coordinates, but given the ecological differences between the two new sympatric species, only B.ommata inhabits Aare lake systems. We therefore assign these historical records tentatively to this species. Despite considerable sampling efforts using electrofishing, minnow traps and nets across different seasons and years, no Barbatula specimens were observed from lakes Thun and Brienz after 2011. Given the very limited number of records, each separated by a decade or more (except for 2001), we assume that B. ommata previously inhabited these lakes but that its population has since declined drastically, likely potentially approaching local extinction. Given the limited historical records of the species from lakes Thun and Brienz and the lack of recent confirmed occurrences, a targeted search for these subpopulations is recommended to clarify their potential persistence in these lakes and assess the species' conservation status. Additionally, only three specimens of B.ommata were recorded in Lake Murten (also known as Lac de Morat) in 2010, two of which were detected during Project Lac and one in NAWA monitoring. The InfoFauna database contains just two further records from this lake, in 1985 and 2001. A similar situation applies to Lake Zug, where there are only four specimens recorded in 2013 during project Lac. Data indicate that B. ommata is present at low abundance in Lakes Murten and Zug. Given the declining habitat quality in these lakes, we recommend systematic monitoring to assess the population dynamics and conservation status of this species. Such monitoring is crucial for developing effective conservation strategies to mitigate further habitat degradation and support the survival of B. ommata in these lakes. Sexual dimorphism.Barbatula ommata shows epidemical tubercles in the pectoral-fin rays of adult males. The tubercles are present mostly on the innermost fourth branched pectoral-fin rays (vs. tubercles absent or not evident in females and juveniles). Difference in pectoral-fin shape is easily noticed in adults, with male showing pointed pectoral-fin tip and enlargement of outermost branched-fin rays (vs. rounded in female, and fin rays similar in width). Urogenital papilla of males small and conical (vs. urogenital in females rounded). Etymology.Barbatula ommata from the Greek ómmata (ὄμματα) for eyes, and is given in reference to the species diagnostic great diameter of its eyes. A noun in apposition. Vernacular name. Lake Stone Loach (English), Seebartgrundel (German), Loche du Lac (French). Ecological notes. Specimens were collected in the littoral zone of lakes. Collected on the shore down to 1.2 m water depth, most abundantly found in small pebbles substrate, and among middle-sized stones (20 cm). Barbatula ommata and Cottus sp. are syntopic and were collected together. Few individuals dissected indicate that the species is primarily insectivorous. Conservation status.Barbatula ommata seems endemic to Switzerland, with its distribution largely confined to lakes within the Aare catchment, including Neuchatel, Biel, Murten, Lucerne, Walen, Zug and Zurich (Figure 3). Despite extensive sampling efforts, the species has not been observed in lakes Thun and Brienz in the past 15 years, despite sporadic historical records. Assessing whether the CALEGARI ET AL.21 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License species is approaching local extinction in these lakes is critical, as such a loss would account for 25% of its overall distribution and one entire subcatchment of the four within its original range. The Extent of Occurrence (EOO) based on the extant range of the species was estimated at 4302 km 2 by the Minimum Convex Polygon (calculated using GeoCAT tool) meeting the IUCN criteria of Endangered (B1: EOO < 5000 km 2 ). The area of occupancy (AOO) based on IUCN methodology (2 2 km grid) of known records was estimated at 132 km 2 meeting the criteria of Endangered (B2: AOO < 500 km 2 ). The AOO for the entire lake area for all lakes where the species occurs was also estimated at 490 km 2 , which represents an overestimation because this species is confined to shallow littoral zones of the lakes (<1.5 m depth) requiring pebbles and small stones substrate, and it is absent from deeper areas as well as large boulders and rock faces. Barbatula ommata has a restricted distribution driven by its specific habitat requirements. Accordingly, we estimate the extent of suitable habitat within the very restricted range for the species at 26.2 km 2 , considering only the shoreline areas of all lakes and excluding deeper zones where the species does not occur. We used a geospatial buffer from the shoreline to make this calculation, assuming all habitat within 50 m of the shoreline was habitable within all lakes where the species has recently been recorded (Zurich, Neuchatel, Murten, Biel, Lucerne, Walen and Zug; note that the median distance from the shoreline for B.ommata records was 13 m). The distribution of B.ommata in Swiss lakes is severely affected by urbanisation. Many areas are impacted by habitat degradation, primarily from urbanisation, pollution and the alteration of littoral zones caused by constructions, harbours, boulders and retaining walls, yet the impact degree of these modifications remains largely unknown. The loss of suitable habitats, already highly restricted (26.2 km 2 ), poses a significant threat to the species, which is confined to shallow littoral zones, absent from deeper lake areas and dependent on pebble and small substrate for survival. This specific habitat has currently a patchy distribution in the lakes that B. ommata occurs in, which further isolates subpopulations within the lakes of this species whose range is already naturally fragmented across lakes. Additionally, the extensive recreational use of the last natural lake shores, particularly in the summer, has potentially caused a continued decline of habitat quality and area of occupancy due to this species having its reproductive activity, feeding areas and nursery grounds in the littoral zones. Climate change is also an important factor that has been negatively impacting cold-water lakes due to the increase of water temperature in the shallow inshore zones in summer, despite it being difficult to measure the extent of impact. Barbatula ommata is naturally confined to large, oligotrophic, clear water lakes and its range is already naturally severely fragmented. Many of these lakes are surrounded by urban areas undergoing significant habitat degradation, with a notable loss of suitable habitat across multiple locations. Subpopulations in lakes Thun, Brienz, Murten, Biel and Zug are very small and have not been observed for over 12 years despite targeted collection efforts. These lakes together represent approximately 50% of the species' distribution within the lake system. The prolonged absence of B.ommata suggests a significant reduction in the AOO of the species and the population is assumed to be severely fragmented. Thus, Barbatula ommata is preliminary assessed as Endangered (EN) based on the criteria B1ab(ii,iii) +B2ab(ii,iii), according to IUCN criteria (IUCN Standards and Petitions Subcommittee, 2022). 3.2 |Statistical morphometric analyses We investigated the populations of the two new species (B.fluvicola and B.ommata) and the most closely related valid species belonging to the Western clade (B.barbatula,B.hispanica,B.leoparda), through PCA. To test whether the populations from new species are conspecific, they were also treated with different symbols in a preliminary analysis. This analysis has not shown segregation between populations within the same species, so each species was here represented under a unique symbol. PCA showed a distinct separation between all species (Figure 14a). PC1 explains 78.8% and PC3 explains 3.9% of the variation between specimens of the species analysed. Five variables had stronger contribution to discriminate both new species B.fluvicola and B.ommata from each other: larger and smaller orbital diameter, respectively; smaller and larger caudal-peduncle width and caudalpeduncle depth, respectively; smaller and larger mental lobe length, respectively; and narrower and wider mouth, respectively. Other variables that have contributed to separate the two new species from remaining congeners are shallower, wider and longer caudal peduncle, and upper-lower lips distance. Orbital diameter, upper-lower lips distance, and dorsal head length and head length loaded more strongly positively, while caudal-peduncle width and caudal-peduncle depth loaded more negatively on PC3. The PCA analysis of morphometric data in populations of Barbatula fluvicola from Rhine drainage in Switzerland, and from Danube drainage in Germany showed an overlap with latter population completely nested in the Switzerland population range, confirming our findings that they represent the same species; PC1 explain 69.5% and PC2 explain 7.7% the variation found (Figure 14b). 3.3 |Molecular analyses and species delimitation A total of 506 new Barbatula coI sequences (317 sequences from the FREDIE project and 189 sequences from this project) were used for the molecular analyses in addition to the available GenBank/BOLD sequences. All sequences from this study were submitted to GenBank (see Data S2 for specimen metadata and GenBank accession numbers). The final coI gene alignment length was 651 base pairs. The ML analyses were conducted by codon partition, as suggested by PartitionFinder. The summarised unrooted ML tree for the entire dataset (AB dataset) is presented in Figure 15a and the rooted ML tree is shown in Data S3. The ML analysis of the AB dataset revealed five major clades within the Asian Barbatula species (Asian clades A–E), three Eastern Europe clades (Eastern clades A–C) and one Western Europe clade 22 CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License (Figure 15 and Data S3) and further showed a deep divergence between Asian and European Barbatula clades. The ML tree of the EB dataset is shown in Data S4.Theresultsof the ASAP, PTP and mPTP species delimitations for the EB dataset ranged from 9 to 26 MOTUs (Table 3and Data S2). The optimal ASAP score was 3.5, yielding three distinct species delimitation scenarios, which resulted in 14, 18 and 22 MOTUs, respectively. Despite the three distinct delimitation scenarios, most MOTUs were consistently recovered. To ensure consistency with morphological assignments and align with the most coherent species delimitation across the European freshwater Barbatula populations (based on an ASAP score of 3.5), we will use the species assignments with 18 MOTUs (Data S4). From the 18 MOTUs recognised, 10 can be currently associated with available nomen: Barbatula b. taurica (Kessler, 1877)+Barbatula b. caucasica (Berg, 1898)(EasternlineageB1),B.zetensis (Eastern lineage C1), B.sturanyi (Eastern lineage C2), B.pironae (Nardo, 1866) (Eastern lineage C5), B.vardarensis (Eastern lineage C8), B.fluvicola (Western lineage W1), B.barbatula (Western lineage W2), B.leoparda (Western lineage W3), B.hispanica (Western lineage W5) and B.ommata (Western lineage W6). The additional eight MOTUs possibly represent distinct species, for which morphological data is needed to describe and validate them. These include Barbatula sp. (Eastern lineage A1, Romania), Barbatula sp. (Eastern lineage A2, Ukraine), Barbatula sp. (Eastern lineage C3, Romania), Barbatula sp. (Eastern lineageC4,ArgesandMuresriversinRomania),Barbatula sp. (Eastern lineage C6, Croatia), Barbatula sp. Eastern lineage C7, Romania), Barbatula sp. (Eastern lineage C9, Germany, Austria, Czech Republic, Sweden) and Barbatula sp. (Western lineage W4, Dordogne, Garonne, Agly, Dourbie rivers in France, Segre river in Spain) (Data S4). In the ML analysis, clades containing both previously recognised subspecies B. caucasica and B. taurica, and B. pironae were recovered as distinct lineages, separate from other valid species and phylogenetically distant from each other (Data S4). Barbatula caucasica and B. taurica were assigned to the same MOTU (Eastern lineage B1). As our phylogeny and species delimitation relied solely on the coI gene, and morphological analyses of these populations were not conducted, a more comprehensive study is needed to clarify their taxonomic status and geographic distribution. Although these species are recognised as valid here, their redescription is out of the scope of our present study. To better investigate the species diversity in the Western Europe clade, and to avoid interference because of the high genetic divergence between Eastern and Western European clades, we conducted a separate species delimitation analysis based on the WB dataset alone (Table 3). The ML tree of the WB dataset is shown in Figure 16 (Data S5 for details). The number of MOTUs delimitated with the three species delimitation methods ranged from 5 to 16. Most individuals previously morphologically assigned to a specific species were grouped together in the molecular analysis. However, PTP analysis with 16 MOTUs showed a clear oversplitting. The mPTP method resulted in six species, while the ASAP method delimitated five and six species, both with the score of 2. We follow the species delimitation obtained with ASAP (best score 2), which recovered 6 MOTUs because is best corroborating our a priori morphological assignments. The main distinction between the two ASAP methods (score 2) results was that Barbatula sp. (Western lineage W4) was not delimitated as an independent species from B.hispanica in the 5 MOTUs solution. The six delimitated species supported by both ASAP and mPTP methods are corroborated by morphological differentiation. Barbatula fluvicola is the sister taxa of a clade including all remaining Western Europe congeners, with B.ommata, sister taxa of the clade formed by B.barbatula (B.hispanica +B.leoparda +Barbatula FIGURE 14 (a) Biplot of first and third principal components analysis of morphometric data in Barbatula barbatula (red exes), B.hispanica (orange crosses), B.leoparda (purple diamonds), B.fluvicola (light blue triangles) and B.ommata (yellow squares). Percent of variation included in PC1 is 80.2% and in PC3 is 3.8%. B =Biplot of first and second principal components analysis of morphometric data in populations of B.fluvicola from Rhine drainage in Switzerland (light blue triangles) and Danube drainage in Germany (dark blue diamonds). Percentage of variation included in PC1 is 69.5% and in PC2 is 7.7%. CALEGARI ET AL.23 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License sp. W4) (Figure 15b). It is noteworthy, otherwise, that the larger group containing B.ommata delimitated by the ASAP and mPTP methods includes a small subclade (recognised by the PTP method) with seven sequences from Ireland, which represent introduced population since Barbatula does not naturally occur in this area. The pairwise distances were compared at the intraspecific and interspecific levels (Table 4). The species discrimination power of DNA barcoding for Western Europe Barbatula species was confirmed by the barcoding gaps. The two new species, B.fluvicola and B.ommata, were clearly distinguished from each other by genetic distances ranging from (2.39% to 3.31%). Barbatula fluvicola is separated from the Western Europe clade species by a genetic distance ranging from 2.71% to 4.46%. Barbatula ommata is separated from the other Western clade congeners by a genetic distance value ranging from 2.15% to 3.69%. 4|DISCUSSION Barbatula barbatula has historically been used to refer to Barbatula populations across Europe, but the lack of a precise type locality and extant type specimens has posed a major challenge to stabilising its nomenclature. To address this, we designated a neotype (Figure 2) from the Lez River (originally the type locality of Barbatula b. quignardi), identifying it as the likely population described by Rondelet (1555), which formed the basis for Linnaeus' (1758) description of Cobitis barbatula. This taxonomic action, in accordance with ICZN guidelines (International Commission on Zoological Nomenclature, 1999), clarifies the identity of the type species B. barbatula, resolves longstanding ambiguity and establishes B. quignardi as a junior synonym of B. barbatula. FIGURE 15 Results of the barcoding portion of the coI gene for Barbatula. (a) Unrooted phylogram of the maximum likelihood analysis of all Barbatula (AB dataset) with the major clades indicated in different colours. (b) Rooted phylogram of the maximum likelihood analysis of the Western Barbatula clade (WB dataset). The major monophyletic groups (W1–W6) were collapsed. Bootstrap support values are shown next to individual branches. 24 CALEGARI ET AL. FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4.1 |Phylogeny and geographical distribution The morphological approach was used to re-describe B.barbatula and describe two new species strengthens species delimitation and classification within the genus. Additionally, our mitochondrial phylogeographic analysis of Barbatula across Europe provides a key basis for future taxonomic investigation (Data S4). We found four well-supported major European Barbatula clades, three in Eastern Europe and one in Western Europe (Figure 15a). Eastern subclades include B.sturanyi,B.vardarensis, B.zetensis,B. caucasica,B. pironae and B. taurica and several lineages indicative of possibly further new species. Within this clade, B. caucasica, B. pironae and B. taurica are not sister species to B.barbatula, supporting them as valid species that require a further diagnose and redescription, which is beyond the scope of this study. The diversity patterns and phylogeographic structure of the Western lineages of Barbatula seem to reflect a shared biogeographic history with other European freshwater fish species, such as Telestes (Salzburger et al., 2003). This shared history was shaped by glaciations, slope mass movements and sedimentation, which caused major landscape rearrangements and river flow reversals. The colonisation of Europe by Barbatula is hypothesised to have occurred through a westward geographic expansion of Mongolian lineages through the Siberia region about 5.6 mya (ˇ Slechtová et al., 2024). This timing is coincident with the increasingly wet climate, which promoted the formation of wetlands pathways that could have facilitated the dispersion from Central Asia to Western European rivers (ˇ Slechtová et al., 2024). This evidence supports the earlier origin of the European Eastern lineages, followed by the diversification of Western European lineages. Barbatula radiation likely began in the Pliocene (5.3 Mya; ˇ Slechtová et al., 2024)during climate cooling and continued through Pleistocene glaciations. Noticeably, four out of the six Barbatula species in the Western European clade show allopatric distributions, with possibly only B. fluvicola and B. ommata found in sympatry. These species have a non-sister relationship despite sharing their geographic range in the upper Rhine, which indicates allopatric speciation followed by range expansion and secondary sympatry. Given their current geographic distributions, where B. ommata has closest relatives in the Rhône, Western Mediterranean and Cantabria coastal rivers (B.barbatula,B. hispanica,B.leoparda,Barbatula sp. W4), we suggest that ancestral populations of the new species likely occurred in allopatry in the paleodrainages Rhône-Aare (B.ommata) and Danube-Alpine Rhine (B. fluvicola) (Figure 17). During the Pliocene (5.3 Mya), the Alpine Rhine was flowing towards the Danube, while the Rhône and historical Aare drained separately northward through the Swiss Plateau and also flowed towards the Danube (Muhar et al., 2019). Topographic changes redirected the Alpine Rhône and later the Aare westward, diverging from their original Danubian flow. At this time the Aare became a tributary to the Rhône. This shift likely isolated Alpine Rhône and historical Aare populations from the Stone Loaches in the Danube system, driving the speciation process in the western portion of Alpine Arc (B. barbatula,B. hispanica,B. leoparda,Barbatula sp. W4, B. ommata). It is probably since this time that the ancestors of B.ommata and its western relatives have been isolated geographically from those of B. fluvicola. The Rhine's retrograde erosion eventually extended its course south to Basel and beyond, capturing the Aare and separating its Stone Loach population from those in the Rhône drainage (Keith et al., 2011; Muhar et al., 2019). Finally, the repeated advance and retreat of the Pleistocene ice shield shaped the Alpine Rhine and headwaters of Aare and Rhône, cutting deep valleys in which the deep pre-Alpine lakes are now situated (Preusser et al., 2010), now reduced remnants of their former much more massive size. As the glaciers finally retreated 160000 years ago, the area that is now Switzerland would have been colonised by B.fluvicola either directly from the Danube or through the Rhine after crossing from the Danube to the Rhine in Southern Germany. Barbatula ommata on the other hand would have survived the glacial maximum in the upper Rhine and/or lowermost Aare portion in what is now Germany and Switzerland and would have followed the retreating glaciers upwards into the Aare system. This existence of large newly formed lakes in the Aare would possibly have TABLE 3 Species delimitation scores analysed for Europe Barbatula lineage (a–c) dataset and Western Barbatula lineage dataset, estimated based on three distinct methods: ASAP, PTP and mPTP. Species delimitation method Eastern European lineage A Eastern European lineage B Eastern European lineage C Western European lineage Total nMOTU Europe Barbatula dataset (718 taxa) ASAP score 3.5 2 1 6 5 14 ASAP score 3.5 2 1 9 6 18 ASAP score 3.5 3 1 9 9 22 mPTP 1 1 5 2 9 PTP 3 1 13 9 26 Western Europe Barbatula dataset (418 taxa) ASAP score 2 n/a n/a n/a 5 5 ASAP score 2 n/a n/a n/a 6 6 mPTP n/a n/a n/a 6 6 PTP n/a n/a n/a 16 16 Abbreviation: n/a, not applicable. CALEGARI ET AL.25 FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FREDIE project (Freshwater Diversity Identification for Europe). We thank Project Lac and Progetto Fiumi for kindly provide material for analyses. We thank David Haberthür, Ruslan Hlushchuk and the medicine faculty facility for the service in generating high quality HRXCT images of the new species. We are very grateful to Anne-Claire Fabre (NMBE) for her help and training with the HRXCT software. We thank Nadine Apperle for helping with the translation of natural history books written in old German. We thank to Roberto Reis, Tiago Carvalho, and Sven Kullander for the productive discussions about ICZN codes and nomenclature. We are grateful to Marcel Häsler and Soraya Villalba for their assistance in the Lab and with the collection database, and Jakob Brodersen and Luiz Jardim de Queiroz for the constructive discussions about ecology and population genetic of fishes from Switzerland. We are grateful to Oliver Selz, Diego Dagani, and Carmela Doenz (FOEN) for their constant support on fish biodiversity studies, and valuable information and discussions about fish diversity and Barbatula populations. We further thank the media offices from University of Bern (Ivo Schmucki, Patrizia Jaeggi), Wyss Academy for Nature (Seta Takur and Cyrill Hess), and Eawag (Claudia Carle) for supporting our educational project straighten science-society knowledge transfer helping in the communications, and the LANAT-3 team project, especially Adrian Aeschlimann, Murielle Neuhaus, and Pia Fehle for being supportive in the development of the present study. We are grateful to the general public for participating in the open survey and helping to select the names of the two new. We also thank the Naturhistorisches Museum Bern for generously providing museum entry tickets as prizes for the campaign. We are grateful for the financial support of the Canton of Bern, FOEN, and Wyss Academy for Nature to develop this study. Open access publishing facilitated by Universitat Bern, as part of the Wiley - Universitat Bern agreement via the Consortium Of Swiss Academic Libraries. ORCID Bárbara B. Calegari https://orcid.org/0000-0001-6335-6854 Jörg Freyhof https://orcid.org/0000-0002-6762-8615 Conor Waldock https://orcid.org/0000-0002-2818-9859 Bernhard Wegscheider https://orcid.org/0000-0002-9000-451X Dario Josi https://orcid.org/0000-0003-4543-4438 Lukas Rüber https://orcid.org/0000-0003-0125-008X Ole Seehausen https://orcid.org/0000-0001-6598-1434 REFERENCES Aitchison, J. (1986). The statistical analysis of compositional data. 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FISH 10958649, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfb.70108 by Museum Für Naturkunde, Wiley Online Library on [04/09/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License