Integrative taxonomy reveals non-native Squalius hybrids in the Iberian Peninsula

249 Integrative taxonomy reveals non-native Squalius hybrids in the Iberian Peninsula Christos Gkenas1* , Sofia Nogueira1* , Manuel Curto2,3 , Diogo Dias1,4 , Diogo Ribeiro1, Rui Rivaes1, Joana Garrido Nogueira2,3 , Manuel Lopes-Lima2,3 , Maria Judite Alves4,5 , Filipe Ribeiro1 1 MARE, Marine and Environmental Sciences Center/ARNET, Aquatic Research Network, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal 2 CIBIO, Research Center in Biodiversity and Genetic Resources, InBIO Associate Laboratory, Vairão Campus, 4485-661 Vairão, Portugal 3 BIOPOLIS, Program in Genomics, Biodiversity and Land Planning, Vairão Campus, 4485-661 Vairão, Portugall 4 cE3c – Centre for Ecology, Evolution and Environmental Changes/Global Change and Sustainability Institute, Faculty of Sciences, University of Lisbon, 1749016 Lisbon, Portugal 5 MUHNAC – National Museum of Natural History and Science, University of Lisbon, 1250-102 Lisbon, Portugal Corresponding author: Christos Gkenas ([email protected]) Copyright: © Christos Gkenas et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The introduction of non-native species poses a critical threat to biodiversity in Mediterranean freshwater ecosystems. This study documents the first record of non-native Squalius hybrids in the Ave River (Iberian Peninsula), combining morphological and molecular data to confirm their presence and assess their potential origin. The specimens exhibit hybrid characteristics, with most morphological and meristic traits overlapping those of both S. cephalus and S. squalus. Almost all individuals analysed showed an admixed genomic background for both species, suggesting a hybrid origin likely within the species’ native range before their introduction into Portugal through human-mediated activities, likely related to recreational fishing. Furthermore, molecular data also supports hybridization with the native S. carolitertii, raising conservation concerns given the negative impacts associated with hybridization with non-native species. By combining data from social media and citizen science platforms, we were able to map the rapid expansion of this invasive chub in the Ave River from 2018 until now. Our findings emphasize the urgent need for population control measures, while enhancing monitoring, genetic studies, and increased public awareness to mitigate the impacts of invasive species on Iberian freshwater biodiversity. Key words: Ave river, chub, hybridization, invasive species, morphology, Portugal, Squalius cephalus, Squalius squalus Introduction The Mediterranean region is considered a freshwater biodiversity hotspot, characterised by high levels of endemic species shaped by its unique palaeogeographic history (Zardoya and Doadrio 1999). However, fish species face severe threats in this region, mainly caused by human pressures including pollution, habitat destruction, fragmentation, and the introduction of non-native species, with the rapid spread of these species in recent decades being particularly concerning (Clavero et al. 2010; Hermoso et al. 2011; Milardi et al. 2022). Addressing this trend Academic editor: Josie South Received: 30 January 2025 Accepted: 15 July 2025 Published: 7 October 2025 Citation: Gkenas C, Nogueira S, Curto M, Dias D, Ribeiro D, Rivaes R, Garrido Nogueira J, Lopes-Lima M, Alves MJ, Ribeiro F (2025) Integrative taxonomy reveals non-native Squalius hybrids in the Iberian Peninsula. In: Anastácio P, Ribeiro F, Chainho P (Eds) Invasions in Aquatic Systems. NeoBiota 102: 249–268. https://doi.org/10.3897/ neobiota.102.148337 NeoBiota 102: 249–268 (2025) DOI: 10.3897/neobiota.102.148337 * These authors contributed equally to this work. Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 250 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius requires the implementation of effective biosecurity strategies supported by active monitoring of new occurrences and research into their dispersal pathways and vectors (Ricciardi et al. 2021). Within the Mediterranean region, the Iberian basins have some of the highest proportions of native species at risk of extinction (Costa et al. 2021), a situation that has been significantly exacerbated by the proliferation of non-native species (Clavero et al. 2013; Anastácio et al. 2019; Valerio et al. 2022). Most of the recently established species in the Iberian Peninsula originate from other parts of Europe (Anastácio et al. 2019). The northeastern Iberian basins are regarded as a key gateway for species introductions from France, facilitating a northeast-to-west spread across both Spanish and Portuguese basins (García-Berthou et al. 2005; Clavero and García-Berthou 2006), although some evidence suggests alternative introduction pathways (Ribeiro and Veríssimo 2014; Garcia-Raventós et al. 2020). There is, however, a consensual understanding that non-native fishes are primarily introduced for recreational fishing purposes, either as bait or as targeted species (Banha et al. 2017a; Anastácio et al. 2019), a trend that continues to grow (Banha et al. 2024). In recent years, technological advancements and increased awareness of non-native species impacts have improved our capacity to monitor new species arrivals. Nevertheless, monitoring aquatic habitats remains challenging due to logistical constraints, including limited accessibility to remote locations, insufficient funding and equipment availability, and a lack of trained personnel. Additionally, the introduction of species that are morphologically similar or indistinguishable from other already established non-native species, such as Carassius auratus (Linnaeus, 1758) and Carassius gibelio (Block, 1782) (Ribeiro et al. 2015) or those resembling native species, presents significant challenges for accurate identification, monitoring, and management (e.g., Aparicio et al. 2013; Pérez-Bielsa et al. 2025). These morphological similarities can delay the identification of newly occurring species, allowing them to spread undetected for extended periods. In this context, species from highly diverse genera including minnows (Artaev et al. 2024), chubs (Buj et al. 2020) and barbels (Rossi et al. 2021), which share ambiguous morphological traits and have taxonomic classifications that remain actively debated, are particularly susceptible to these identification and monitoring challenges. This is the case of the genus Squalius Bonaparte, 1837, which is present across European watersheds and comprises more than 50 species, many of which are endemic to specific drainages in the Meridional peninsulas (Kotellat and Freyhof 2007; Perea et al. 2020). Most of these species share morphological features and exhibit overlapping meristic traits. In the Iberian Peninsula, thirteen species of Squalius have been identified (Mendes et al. 2024), many of which have been described mainly based on molecular data, with subtle morphological differences (e.g., Doadrio and Carmona 2006; Doadrio et al. 2007). Until now, no non-native species from this genus has been recorded in Portugal. However, recent genetic analyses have identified a chub population of Squalius cephalus (Linnaeus, 1758) and evidence of its hybridization with the endemic Squalius laietanus Doadrio, Kottelat and de Sostoa, 2007 in Spain (Pérez-Bielsa et al. 2025). Furthermore, eDNA data from the Ave River detected the presence of Squalius squalus (Bonaparte, 1837), which needs to be confirmed (Curto et al. 2025). This study examines the occurrence of non-native Squalius hybrids in the Ave River using morphological and molecular analyses. Additionally, we used data mined from social media forum groups to map recent occurrences and to construct their current potential distribution in Portugal. 251 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius Materials and methods Study area This study was conducted in the Ave River, northern Portugal, which hosts a single native Squalius species: the Northern Iberian chub, Squalius carolitertii (Doadrio, 1988). This species rarely exceeds 25 cm, has a moderately compressed body, rounded snout with slightly subterminal mouth, and silvery-olive scales without dark margins. Its paired, anal, and caudal fins are hyaline to pale amber with no vivid pigmentation (Collares-Pereira et al. 2021). In contrast, non-native chubs in Portuguese basins, documented through angler photos and iNaturalist records, exceed 40 cm, have bulkier heads with terminal mouths, display orange-red or black ventral and anal fins, and show scales with dark edges forming a reticulate pattern. (Kottelat and Freyhof 2007). These differences in size, fin coloration, and scale pattern provide clear criteria for anglers and citizens to distinguish native S. carolitertii from non-native chubs. Between 2017 and 2025, we collected 37 unidentified chubs, using electrofishing in the Ave River (Table 1). The first specimen (FRISK3481) was captured in 2017 during a regular fish monitoring campaign. Upon examination, there was a suspicion that the specimen could be an unreported non-native species to Portuguese freshwaters and thus a tissue sample was kept for genetic analysis. For the same reason, in 2020, tissue samples were collected from seven additional individuals (PEI0456–PEI0462). The remaining 29 individuals were captured during subsequent monitoring campaigns between 2024 and 2025 at four distinct locations along the Ave River (Table 1). Fin clips were extracted from these specimens and preserved in absolute ethanol for genetic analyses. For these, the whole individuals were kept for morphological analysis. The one specimen captured in 2024 was transported frozen to the laboratory and stored at -20 °C for posterior morphological identification. The 28 specimens captured in 2025 were euthanized in the field using clove oil and immediately fixed in 10% formalin. Following fixation for at least three days, these specimens were gradually transitioned to a 70% ethanol solution for long-term preservation, involving exposure to progressively higher ethanol concentrations (30% for two days, followed by 50% for three days). Additionally, to serve as reference for the genetic analysis, tissue samples from six S. cephalus individuals from the native range (50°01'55.6"N, 14°59'35.0"E), captured in 2022, and four from S. carolitertii, captured in 2017 in the Ave River, were added. Morphological analysis In the laboratory, the specimens were photographed, and species identification was carried out using general identification keys (Doadrio and Carmona 2006; Kottelat and Freyhof 2007). Morphological measurements were conducted from point to point to the nearest 0.1 mm with vernier calipers on the left side of the specimen (Suppl. material 1), following the methodology described by Bogutskaya and Zupanič (1999; 2010). The standard length was measured from the tip of the snout to the end of the hypural complex. The length of the caudal peduncle was measured from the posterior edge of the base of the last anal fin ray to the end of the hypural complex, at the mid-height of the caudal fin base. Both head length and interorbital width were measured including the skin fold. Post-dorsal length was measured from the insertion point of the dorsal fin to the end of the hypural complex. The total lateral-line scale count included all pored scales, extending from the first scale 252 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius just posterior to the post temporal bone to the most posterior scale located at the base of the caudal-fin rays. Fin rays were counted separately as simple and branched rays (Bogutskaya and Zupanič 2010; Suppl. material 1). The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins were noted as “1½”. Prior to statistical analysis, all morphometric variables were standardized to remove the effects of size following Kottelat and Freyhof (2007). Head-related measurements were expressed as proportions of head length (HL), while all remaining body measurements were expressed as proportions of standard length (SL) or body depth. Differences in morphometric and meristic characteristics between the studied groups were assessed using independent two-sample (Student’s) t-test, after confirming normality with Shapiro–Wilk tests (Zar 1996). All data processing and analyses were performed in R version 4.4.3 (R Development Core Team 2025). Specimens and tissues were deposited in the zoological collections “Museu Bocage” of the Museu Nacional de História Natural e da Ciência, Universidade de Lisboa, Portugal (MNHNC-MB05-004006 – MB05-004015) (Suppl. material 2). Genetic analysis DNA was extracted from fin clips or muscle tissue preserved in absolute ethanol using commercial kits. More specifically, the seven samples collected in 2020 were processed using EZ-10 Genomic DNA Kit (Bio Basic, Canada), while the remaining were processed using the E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek, Norcross, GA, USA). Both approaches followed the manufacturer’s protocols. DNA quality was assessed through 0.8% agarose gel electrophoresis. Molecular assessment of specimen identity was based on variation at three loci. The mitochondrial cytochrome b (cyt b) gene was amplified through the primer set Glu-F and Thr-R (amplicon ~1140 bp, Zardoya and Doadrio 1998). Two nuclear genes were also targeted: early growth response 2b (egr 2b, ~747 bp; Waap et al. 2011) and beta-actin (actb, ~933 bp; Robalo et al. 2006). PCR reactions were carried out in a 10 µl volume containing 5 µl of MyTaq Mix (Meridian Bioscience, Cincinnati, Ohio, USA) and 0.4 µl of each primer (10 µM). The following temperature profile was implemented: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 1 min, 55 °C for 1 min (50 °C in the case of cyt b) and 72 °C for 1 min, and a final extension at 72 °C for 10 min. PCR success was evaluated through 2% agarose gel electrophoresis. The resulting amplicons were sequenced in both directions using Sanger sequencing, implemented by the molecular analysis services from STAB Vida (Caparica, Portugal). The obtained chromatograms were manually aligned using Geneious Prime (Biomatters Ltd., New Zealand, v. 2025.1.2). For each locus, the sequences generated in this study were joined to publicly available sequences in NCBI for the Table 1. Details of Squalius specimens collected for genetic analysis, including sample codes, number of individuals (n), geographic coordinates (latitude and longitude), and year of collection. Samples marked with an asterisk (*) were also examined for morphological and meristic characteristics. Codes Samples (n) Coordinates Year FRISK3481 1 41°21'05.0"N, 8°40'54.6"W 2017 PEI0456–PEI0462 7 41°20'48.9"N, 8°35'48.0"W 2020 Scep_1_13* 1 41°22'22.7"N, 8°42'07.6"W 2024 Squa1-Squa10* 10 41°24'43.8"N, 8°22'46.2"W 2025 Squa11-Squa13* 3 41°28'18.1"N, 8°20'48.0"W 2025 Squa14-Squa28* 15 41°26'57.1"N, 8°21'00.2"W 2025 253 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius genus Squalius (Suppl. material 3). Up to 10 sequences per each species were downloaded with care to cover multiple drainages for widespread taxa; otherwise, 10 random sequences were downloaded. Additionally, three sequences from the genus Iberochondrostoma Robalo, Almada, Levy & Doadrio, 2007 were added as outgroups. Sequences were aligned using AliView (Larsson 2014) implementing the MUSCLE algorithm. For the nuclear markers (egr 2b and actb) haplotypes were phased in DnaSP v. 5.10.1 (Librado and Rozas 2009). Genetrees for each marker were estimated using the maximum likelihood method implemented in IQtree (Nguyen et al. 2015). The same program was used to select for the best substitution model considering partitions based on codon position. Node support was obtained through 1000 fast bootstraps. IQtree was run using the web server W-IQ-TREE (Trifinopoulos et al. 2016). The obtained tree was visualised and customised using FigTree ver. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Social media and citizen science data collection The use of social media information about invasive fishes has proven valuable for detecting and identifying new invasive fish (Ribeiro and Veríssimo 2014; Kalous et al. 2018), describing spatial invasion patterns (Banha et al. 2017b), and updating the distribution of recently arrived invasive species (Martelo et al. 2021). To update the distribution of alien chubs in the Ave River, extensive data collection was conducted using various sources of information as recommended by Tricarico (2022), including Facebook and citizen science platforms. Publicly available social media posts related to fishing for individuals belonging to the genus Squalius were collected from Facebook, a popular platform among anglers in Portugal (see Gago et al. 2016; Banha et al. 2017b). The data collection process adhered strictly to ethical guidelines for using social media in fisheries research, ensuring that no personal information was recorded and that no original user content was shared publicly without permission (Monkman et al. 2018). Searches were conducted within fishing-focused groups where catches predominantly occur in Portuguese waters following the guidelines described in Chowdhury et al. (2024). The time window of our search was from July 2019 (the creation date of the oldest fishing group) to December 2024. Keywords used to identify relevant posts included the species’ common name and its variants, such as “chub”, “escalo”, “leucisco”, “chevesne” and the nickname “trutas do Ave”. Online searches were also conducted on BioDiversity4All (https://www.biodiversity4all.org/), a Portuguese biodiversity citizen science platform connected to the international project iNaturalist. Species identification was primarily based on the visual examination of photographs accompanying the posts, using criteria established by Gago et al. (2016). Only posts explicitly related to recreational fishing for fish belonging to the genus Squalius were analysed. These posts featured content such as videos and photographs of the fish specimens after being caught. For each post, the primary objective was to extract information on the date and location of the catch. Publications lacking a photographic record of the specimen were not considered for presence records. All posts were manually reviewed to confirm species identification and verify catch locations. Where possible, the background of the photographs was cross-referenced with satellite imagery available on Google Street View to increase the accuracy of the location data (following the georeferencing steps on Chowdhury et al. 2024). The information collected was compiled into a dataset listing locations and dates of the catches, serving as the basis for further analysis. 254 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius Results Morphological analysis After the visual examination of the 29 specimens, 10 were identified as S. cephalus and 19 as S. squalus (Bonaparte, 1837). The S. cephalus individuals had standard lengths (SL) of 107–169 mm and body masses of 20.5–87 g. Fig. 1 illustrates two S. cephalus individuals: the specimen in Fig. 1a (S_cep_1_13), collected in 2024, and the specimen in Fig. 1b (Squa13), collected in 2025. Both individuals (Fig. 1a, b) were streamlined, laterally compressed, and fusiform, lacking a ventral keel anterior to the anus. Each displayed a pointed head, a slightly arched dorsal profile, and a more convex ventral profile, forming a clear head-trunk discontinuity. Flanks showed a reticulate pattern from black pigment along scale margins. Ventral and anal fins were vivid orange-red in Fig. 1a but less intensely coloured in Fig. 1b, which showed only red-orange pigments in the ventral and pelvic fins. a) b) c) Figure 1. Representative hybrid specimens between Squalius cephalus and S. squalus captured in the Ave River, illustrating the observed morphological continuum. a. hybrid individual with predominantly Squalius cephalus-like morphological traits (169 mm total length, 87.0 g); b. hybrid individual with predominantly Squalius cephalus-like morphological traits (135 mm total length, 41.9 g); c. hybrid exhibiting more Squalius squalus-like traits (125 mm total length, 30.1 g). All three individuals were confirmed as hybrids by mitochondrial and nuclear markers and have been deposited in the Museu Bocage collection, Museu Nacional de História Natural e da Ciência (University of Lisbon, Portugal), under accession numbers MB05-004015, MB05-004008 and MB05-004012, respectively. 255 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius The S. squalus specimens ranged from 46–134 mm in SL and weighed between 1.27 and 40.12 g. Fig. 1c shows a representative S. squalus (Squa19), which exhibited an elongated body lacking the pronounced dorsal discontinuity characteristic of S. cephalus. A row of dense black pigment dots along the outer margins of the scales on the back and flanks produced a regular reticulate pattern. Body coloration ranged from bronze to brown, while the ventral and anal fins were typically black to hyaline, without the distinctive red pigmentation seen in S. cephalus. Although all 39 morphometric variables exhibited overlapping values between S. cephalus and S. squalus, a subset of 12 traits was found to significantly differentiate the body shape and proportional anatomy of the two species (Suppl. material 1). Squalius cephalus exhibited a significantly longer postorbital distance (47.3–51.7% HL vs. 41.9–49.1% HL; p < 0.001) compared to S. squalus. The head morphology further differed with S. cephalus having greater head length relative to standard length (24.9–27.9% SL vs. 24.2–26.8% SL; p < 0.05) and greater head length as a proportion of predorsal distance (45.7–52.2% vs. 44.7–48.9%; p < 0.05). Squalius cephalus exhibited a significantly deeper body profile at the dorsal fin origin (23.4–26.2% SL vs. 20.6–25.2% SL; p < 0.01) and a greater distance between the pectoral and pelvic fin origins (25.2–29.9% SL vs. 23.7–29.6% SL; p < 0.05). In contrast, S. squalus displayed a thicker caudal peduncle relative to body depth (40.0–46.4% vs. 35.3–43.2%; p < 0.01). Regarding fin morphology, S. squalus exhibited significantly greater dorsal and anal fin depths relative to body depth (dorsal fin: 80.1–95.9% vs. 78.2–89.2%, p < 0.01; anal fin: 63.5–89.9% vs. 51.1–73.8%, p < 0.01), as well as a larger anal fin depth relative to standard length (15.5–19.1% SL vs. 12.8–17.6% SL; p < 0.05). Squalius squalus had significantly larger eyes relative to both standard length and head length (6.2–8.4% SL vs. 4.9–6.9% SL; 23.3–29.9% HL vs. 19.8–26.8% HL; p < 0.01 for both). It also exhibited greater head depth at the nape compared to S. cephalus (68.3–79.3% HL vs. 67.4–75.2% HL; p < 0.01). The number of branched rays in the anal fin differed significantly, with S. squalus showing mostly 9½ rays (rarely 8½) and S. cephalus 8½ (Suppl. material 1). Other meristic traits did not differ significantly. Genetic analysis Sequence information (in GenBank as PV686259–PV686389) was successfully retrieved for all samples for cyt b and egr 2b, while for actb sequencing failed for two individuals (Table 2, Suppl. material 2). These were added into a final alignment containing additional 217, 44, and 62 sequences from 37, 9, and 11 Squalius species for cyt b, egr 2b, and actb, respectively. The final matrix of the cyt b sequences consisted of 1,156 characters, containing 453 variable characters with 401 parsimony informative sites. The alignments of egr 2b and actb genes comprised 638 and 859 characters, respectively, with 20 and 69 variable sites, of which 17 and 64 were informative. For cyt b, the samples of the unidentified Squalius clustered into three groups with relatively high support values (>80%). Six individuals clustered with S. cephalus, one with the group containing all S. carolitertii sequences, while the remaining individuals grouped with S. squalus (Fig. 2). This last group also includes the Adriatic-Hellenic Squalius clade, comprising S. zrmanjae Karaman, 1928, S. lucumis (Bianco, 1983) and S. prespensis (Fowler, 1977). Unknown Squalius sequences for the nuclear genes were also clustered into three groups (Fig. 2), the main difference being that the S. squalus group forms a polytomy at the 256 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius base of S. cephalus and S. carolitertii for egr 2b, and S. cephalus is paraphyletic to S. squalus for actb. Furthermore, in both genetrees S. carolitertii sequences do not form a monophyletic group being intermingled with other Iberian Squalius Table 2. Summary of morphological and molecular results. Morphological species identification was based on identification keys (Doadrio and Carmona 2006; Kottelat and Freyhof 2007). Measurements were rounded to the nearest 0.1 mm with vernier callipers. Molecular analyses include results for the mitochondrial cytochrome b gene (“cytb”; amplicon ~1140 bp, Zardoya and Doadrio 1998), and haplotypes, labelled as “H1” and “H2”, for the two nuclear genes, the early growth response 2b gene (“egr 2b”; ~747 bp, Waap et al. 2011), and β-actin gene (“Beta actin” ; ~933 bp, Robalo et al. 2006). Sample code First screening Origin Morphological ID cytb egr 2b Beta actin Molecular ID H1 H2 H1 H2 Squa1 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus Squa2 Unknown Squalius sp. Ave River S. squalus S. squalus S. cephalus S. cephalus S. cephalus S. cephalus S. squalus x cephalus Squa3 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. cephalus Fail Fail S. squalus x cephalus Squa4 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus Squa5 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus Squa6 Unknown Squalius sp. Ave River S. squalus S. cephalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus Squa7 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus Squa8 Unknown Squalius sp. Ave River S. squalus S. squalus S. cephalus S. cephalus S. squalus S. squalus S. squalus x cephalus Squa9 Unknown Squalius sp. Ave River S. squalus S. squalus S. cephalus S. cephalus S. squalus S. squalus S. squalus x cephalus Squa10 Unknown Squalius sp. Ave River S. cephalus S. cephalus S. cephalus S. cephalus S. squalus S. cephalus S. squalus x cephalus Squa11 Unknown Squalius sp. Ave River S. squalus S. squalus S. cephalus Iberian lineage S. squalus Iberian lineage S. squalus x cephalus x carolitertii Squa12 Unknown Squalius sp. Ave River S. squalus S. cephalus S. squalus S. cephalus S. squalus S. cephalus S. squalus x cephalus Squa13 Unknown Squalius sp. Ave River S. cephalus S. squalus S. squalus S. squalus S. cephalus S. cephalus S. squalus x cephalus Squa14 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus Squa15 Unknown Squalius sp. Ave River S. cephalus S. squalus S. squalus S. cephalus S. cephalus S. cephalus S. squalus x cephalus Squa16 Unknown Squalius sp. Ave River S. cephalus S. carolitertii S. cephalus S. cephalus S. squalus S. cephalus S. squalus x cephalus x carolitertii Squa17 Unknown Squalius sp. Ave River S. cephalus S. cephalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus Squa18 Unknown Squalius sp. Ave River S. cephalus S. cephalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus Squa19 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. cephalus S. squalus S. cephalus S. squalus x cephalus Squa20 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus Squa21 Unknown Squalius sp. Ave River S. cephalus S. squalus S. squalus S. cephalus S. squalus S. cephalus S. squalus x cephalus Squa22 Unknown Squalius sp. Ave River S. squalus S. squalus S. cephalus S. cephalus S. squalus S. squalus S. squalus x cephalus Squa23 Unknown Squalius sp. Ave River S. cephalus S. carolitertii S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus x carolitertii Squa24 Unknown Squalius sp. Ave River S. squalus S. cephalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus Squa25 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus Squa26 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. cephalus S. squalus S. cephalus S. squalus x cephalus Squa27 Unknown Squalius sp. Ave River S. squalus S. squalus S. squalus S. squalus Fail Fail S. squalus Squa28 Unknown Squalius sp. Ave River S. cephalus S. cephalus S. cephalus S. cephalus S. squalus S. cephalus S. squalus x cephalus S_cep__1_13 Unknown Squalius sp. Ave River S. cephalus S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus FRISK3481 Unknown Squalius sp. Ave River Not available S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus PEI0456 Unknown Squalius sp. Ave River Not available S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus PEI0457 Unknown Squalius sp. Ave River Not available S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus PEI0458 Unknown Squalius sp. Ave River Not available S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus PEI0459 Unknown Squalius sp. Ave River Not available S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus PEI0460 Unknown Squalius sp. Ave River Not available S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus PEI0461 Unknown Squalius sp. Ave River Not available S. squalus S. squalus S. squalus Fail Fail S. squalus FRISK481 S. carolitertii Ave River (native) Not available S. carolitertii S. carolitertii S. carolitertii Fail Fail S. carolitertii FRISK 477 S. carolitertii Ave River (native) Not available S. carolitertii S. carolitertii S. carolitertii Fail Fail S. carolitertii FRISK 478 S. carolitertii Ave River (native) Not available S. carolitertii S. carolitertii S. carolitertii Fail Fail S. carolitertii FRISK 480 S. carolitertii Ave River (native) Not available S. carolitertii S. carolitertii S. carolitertii Fail Fail S. carolitertii SC133 5L S. cephalus Czechia (native) Not available Fail S. cephalus S. cephalus Fail Fail S. cephalus SC73 5L S. cephalus Czechia (native) Not available Fail S. cephalus S. cephalus S. cephalus S. cephalus S. cephalus SC77 5L S. cephalus Czechia (native) Not available S. cephalus S. cephalus S. cephalus S. cephalus S. cephalus S. cephalus SC82 5L S. cephalus Czechia (native) Not available S. cephalus S. cephalus S. cephalus Fail Fail S. cephalus SC67 5L S. cephalus Czechia (native) Not available S. cephalus S. cephalus S. cephalus Fail Fail S. cephalus SC72 5L S. cephalus Czechia (native) Not available Fail S. cephalus S. cephalus Fail Fail S. cephalus 257 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius Figure 2. Reconstructed phylogeny of the cytochrome b (cyt b), early growth response 2b (egr 2b), and beta-actin (actb) sequences of Squalius samples included in present study together with sequences available in NCBI. The new sequences were colored based on the groupings they form: red S. carolitertii / Iberian Squalius lineage, blue S. squalus, and green S. cephalus. Bootstrap values: >70% >80% >90% 0.03 S. torgalensis S. cephalus KU302640 S. illyricusOP728012 S. platyceps S. carolitertii AJ69845 S. illyricus AJ251094 S. illyricus MN166114 S. tenellus S. lepidusAJ252812 Squa23_Ap25 S. carolitertiiFRISK481 S. orientalis; S. turcicus Squa6_Ap25 S. cephalus MG8067701 S. vardarensis MK482045 S. cephalus, S. orpheus Remaining unknown Squalius S. laietanus S. orpheus S. lepidus S. pursakensis S. cephalus S. cephalus KU302635 S. fellowesii OP7228023 S. carolitertii FRISK477 S. irideus S. cephalus MN166129 S. svallize S. squalus OP728021 S. cephalus PP928778 S. pyrenaicus; S. alburnoides S. malacitanus S. carolitertii FRISK480 Squa17_Ap25 S. lepidus Squa28_Ap25 S. zrmanjae S. aphipsi S. carolitertii PP928777 S. illyricus MG806702 Squa24_Ap25 S. aradensis S. carolitertii AJ698452 S. illyricusOR791606 S. illyricus HM560183 S. cii S. carolitertiiAJ698453 S. illyricusOR791605 Squa12_Ap25 S. vardarensis S. microlepis S. squalus KU302634 S. cephalus MN166128 S. illyricus MN166111 I. lemmingii OR381970 Squa16_Ap25 Squa10_Ap25 S. berakOQ797999 S. valentinus S. carolitertii S. squalus PP928780 S. pamvoticus S. lepidus I. lusitanicumKU368953 S. illyricus OR791604 S. squalus KU302627 S. squalus JQ652368 S. peloponensis S. cephalus EU791877 S. castellanus S. cephalus KU302632 S. agdamicusOQ798019 S. squalus, S. zrmanjae S. illyricus HM560184 S. squalus, S. lucumonis S. keadicus S. cephalus OP728027 S. carolitertiiMT817384 S. carolitertii FRIS478 S. lucumonis S. cephalus SC675L Squa18_Ap25 S. svallize S. prespensis Squaliussqualus clade Squaliuscephalusclade Squaliuscaroliteri clade 0.002 Squa19_H1 I. lusitanicum KC836287.1_H2 S. cephalus SC825L_H2 Squa4_H2 Squa20_H1 PEI_458_H2 Squa16_H1 S. cephalus SC675L_2L_H2 Frisk3481_H1 S. cephalusJN413311.1_H2 PEI_459_H1 Squa7_H2 Squa14_H1 S. valentinus I. lemmingi Squa8_H2 S. cephalus SC775L_H2 PEI_462_H2 Squa12_H2 S. pyrenaicus; S. castellanus Squa28_H2 Squa19_H2 Squa15_H1 S. cephalusSC133_5L_Dyje_Breclav_H2 S. cephalus SC133_5L_Dyje_Breclav_H1 S. squalus JASFWY011386779_H2 PEI_456_H1 Squa7_H1 S. pyrenaicus JN413320.1_H2 Squa12_H1 S. cephalus SC775L_H1 S. carolitertii MT051823.1_H1 Squa6_H1 Squa22_H2 S. cephalus SC675L_H2 S. carolitertii MT051824.1_H1 S. cephalus SC825L_H1 S. carolitertii MT051823.1_H2 Squa1_H2 Squa14_H2 S. carolitertii Squa11_H2 Squa17_H2 Squa3_H1 PEI_457_H2 S. carolitertii Squa28_H1 Squa4_H1 Squa2_H2 Squa5_H1 Squa6_H2 PEI_458_H1 S. malacitanus; S. pyrenaicus; S. valentinus Squa27_H2 PEI_459_H2 S. carolitertii; S. pyrenaicus S. pyrenaicus JN413320.1_H1 S. pyrenaicus KC836325.1_H1 S. squalus JASFWY011386779_H1 Squa2_H1 S. carolitertii JN413314.1_H1 Squa22_H1 Squa25_H1 S_cep__1_13_H2 Squa1_H1 Squa13_H2 Squa26_H2 S. cephalusSC735L_H1 Squa25_H2 Squa21_H1 Squa5_H2 S. cephalusSC735L_H2 Squa21_H2 S. cephalus SC675L_H1 Squa9_H2 S. malacitanusMT051748.1_H2 Squa8_H1 Squa16_H2 S. cephalusJN413311.1_H1 Squa13_H1 S. carolitertii MT051840.1_H1 S. malacitanusMT051747.1_H2 Squa23_H2 Squa17_H1 Squa24_H2 Squa23_H1 S. torgalensis; S. valentinus S. pyrenaicus Squa18_H2 Squa9_H1 I. lusitanicum KC836287.1_H1 Squa15_H2 S. carolitertii MT051824.1_H2 Squa18_H1 PEI_457_H1 PEI_456_H2 Frisk3481_H2 Squa24_H1 Squa10_H1 Squa3_H2 PEI_460_H1 PEI_460_H2 S_cep__1_13_H1 S. cephalus Squa10_H2 S. cephalus SC675L_2L_H1 PEI_462_H1 S. malacitanusMT051747.1_H1 Squa11_H1 Squa26_H1 S. pyrenaicus MT051791.1_H1 S. carolitertii MT051818.1_H1 Squa27_H1 Squa20_H2 Squalius squalus clade Squalius cephalusclade Iberian Squali clade 0.003 S. carolitertii Squa28_H2 S. pyrenaicus Squa24_H2 Squa13_H1 S. alburnaides S. aradensis; S. torgalensis Squa6_H2 I._lusitanicum_DQ061941.1_H1 Frisk3481_H2 S. squalus Squa19_H2 S. cephalus_SC735L_H1 S. cephalus_OX439247_H2 S_cep_1_H2 S. carolitertii_MT051720.1_H2 Squa26_H2 Squa2_H2 Squa13_H2 PEI0460_H2 I._lusitanicum_DQ061941.1_H2 Squa17_H2 A. hispanica S. cephalus_OX439247_H1 S. malacitanus PEI0457_H2 PEI0459_H2 Squa11_H2 Squa10_H2 S. pyrenaicus; S. castellanus Squa15_H2 Squa15_H1 S. idus_DQ061947.1_H1 Remaining unknown Squalus Squa21_H2 Squa7_H2 Squa12_H2 S. cephalus_SC675L_H1 S. carolitertii S. valentinus S. carolitertii S. cephalus Squa16_H2 S. pyrenaicus Squa2_H1 S. pyrenaicus S. idus_DQ061947.1_H2 I._lemmingii_DQ061940.1_H2 S. cephalus_SC675L_H2 S. pyrenaicus S. malacitanus I._lemmingii_DQ061940.1_H1 Squaliussqualus clade Squalius cephalus clade Iberian Squaliusclade cytb egr2b actb species. 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Journal of Molecular Evolution 49(2): 227–237. https://doi.org/10.1007/ PL00006545 Supplementary material 1 Results of Student’s t-tests comparing morphometric and meristic variables between hybrid specimens from the Ave River with predominantly Squalius cephalus-like traits and those with predominantly Squalius squalus-like traits Authors: Christos Gkenas, Sofia Nogueira, Manuel Curto, Diogo Dias, Diogo Ribeiro, Rui Rivaes, Joana Garrido Nogueira, Manuel Lopes-Lima, Maria Judite Alves, Filipe Ribeiro Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.148337.suppl1 Supplementary material 2 Voucher specimen information and GenBank accession numbers Authors: Christos Gkenas, Sofia Nogueira, Manuel Curto, Diogo Dias, Diogo Ribeiro, Rui Rivaes, Joana Garrido Nogueira, Manuel Lopes-Lima, Maria Judite Alves, Filipe Ribeiro Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.148337.suppl2 268 NeoBiota 102: 249–268 (2025), DOI: 10.3897/neobiota.102.148337 Christos Gkenas et al.: Integrative taxonomy identifies non-native Squalius Supplementary material 3 Sequences downloaded from NCBI to complement molecular analysis Authors: Christos Gkenas, Sofia Nogueira, Manuel Curto, Diogo Dias, Diogo Ribeiro, Rui Rivaes, Joana Garrido Nogueira, Manuel Lopes-Lima, Maria Judite Alves, Filipe Ribeiro Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.148337.suppl3 Supplementary material 4 Location of records for the non-native Squalius hybrids in the Ave River, derived from social media and citizen science platforms Authors: Christos Gkenas, Sofia Nogueira, Manuel Curto, Diogo Dias, Diogo Ribeiro, Rui Rivaes, Joana Garrido Nogueira, Manuel Lopes-Lima, Maria Judite Alves, Filipe Ribeiro Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.148337.suppl4