Genetic distance and phylogenetic analyse
Multiple alignments of DNA sequences were conducted using Clustal X (Thompson et al., 1997), and manually checked with SEAVIEW (Galtier et al., 1996). Haplotypes collapsed in ALTER (Glezpeña et al., 2010) were used for estimation of phylogenetic trees. The Kimura’s 2‐parameter (K2P) genetic distances were cuculated using MEGA X (Kumar et al., 2018) for the 57 species.
Based on the sequences of Cyt b gene, phylogenetic relationships of 7 Cultrinae species (Culter erythropterus , Culter mongolicus , Culter dabryi , Culter alburnus ,Hemiculter leucisculus , Hemiculter bleekeri ,Pseudolaubuca sinensis ) were reconstructed using Bayesian inference (BI) and maximum likelihood (ML) approaches. We selected 4 species of Xenocypridinae as the outgroup taxa (Xenocypris argentea MH797098, Xenocypris davidi MH797100,Distoechodon hupeinensis MH797101, Pseudobrama simoniMH797105). The best-fit model (GTR+F+G4) of nucleotide substitution for the data was identified by ModelFinder (Kalyaanamoorthy et al., 2017) using the Corrected Akaike Information Criterion implemented in PhyloSuite platform (Zhang et al., 2020). BI and ML analysis were performed with MrBayes v3.2.7 (Ronquist et al., 2012) and IQ-TREE 1.5 (Nguyen et al., 2015), respectively, implemented in the PhyloSuite platform (Zhang et al., 2020). In BI, four simultaneous Markov Chain Monte Carlo (MCMC) were run for 6 million generations sampling every 100 generations, and using a burn-in rate of 25%. In ML analysis, we evaluated the node support by 1000 ultrafast bootstrap replicates (UFBS).
Stable isotope laboratory analysis
Subsamples of muscle tissue (c. 200-300 µg dry mass) were transferred into tin capsules and analysed for carbon and nitrogen stable isotopes using Delta V Advantage mass spectrometer coupled to Flash 2000 HT elemental analyser (Thermo Fisher Scientific, Waltham, MA, USA). Stable isotope ratios were expressed in delta (δ ) notation in parts per thousand or per mil (‰) as deviations from a standard: δ X (‰) = [(Rsample / Rstandard) -1] × 1000, where X is 13C or 15N, Rsample and Rstandard are the13C/12C or15N/14N ratios of the sample and standard, respectively. The standard reference materials Vienna-Pee Dee Belemnite limestone (V-PDB) for carbon and atmospheric N2 for nitrogen (Fry, 1999; Mendoza-Carranza et al., 2010; Riccialdelli et al., 2017). USGS40, USGS41, USGS65, USGS41A, UREA-Thermo and IAEA-600 were used as internal reference standards and interspersed in the samples. The analytical precision (quantified via repeated analysis of internal reference standards) was ± 0.2‰ for bothδ 13C and δ 15N.