Samples of C. brachygnathus were collected from 12 different sampling sites in the Yangtze River Basin from August 2019 to August 2020 (Each sample site was more than 30 individual). From downstream to upstream, these were Taihu Lake (TH), Chaohu Lake (AHCH), Poyanghu Lake (PYH), Dongtinghu Lake (DTH), Changhu Lake (CH), Sandouping (SDP), TaipingXi (TPX), Xiangxi River (XXH), Xiaojiang River (XJ), Wanzhou (WZ), Fengdu (FD), and Fuling (FL). TH and AHCH are affiliated lakes in the lower reaches of the Yangtze River; PYH, DTH, and CH are affiliated lakes in the middle reaches of the Yangtze River, the original distribution of C. brachygnathus in the Yangtze River Basin. SDP is located between the Gezhouba Dam and the Three Gorges Dam; TPX, XXH, XJ, WZ, FD, and FL belong to the Three Gorges Reservoir, of which XXH and XJ are tributaries of the Three Gorges Reservoir, the invasion region of C. brachygnathus in the Yangtze River Basin (Figure 1). Through our investigations, it was found that C. brachygnathus was scarce in the river section above Fuling in the Three Gorges Reservoir, where is still flowing water habitat. Therefore, these 12 sampling sites basically covered the distribution range of C. brachygnathus in the Yangtze River Basin. Species identification mainly refers to Fauna Sinica Osteichthyes Cypriniformes II (Chen, 1998).

After the fish samples were collected, tissues from the dorsal muscles were cut and stored in 95% alcohol at −20°C in a centrifuge tube. Approximately 50 mg of muscles were used to extract genomic DNA using the animal tissue genomic DNA extraction kit of Chengdu Forge Biotechnology Co., Ltd.

The amplification primers for the mitochondrial DNA Cyt b fragment were L14724 5′-GAC TTG AAA AAC CAC CGT TG-3′ (forward) and H15915 5′-CTC CGA TCT CCG GAT TAC AAG AC-3′ (reverse) (Xiao et al., 2001). The amplification primers for the mitochondrial DNA D-Loop fragment were DF1 5′-CTA ACT CCC AAA GCT AGA ATT CT-3′ (forward) and DR2 5′-ATC TTA GCA TCT TCA GTG-3′ (reverse) (Tang et al., 2007). The total volume for the Polymerase Chain Reaction (PCR) was 30 μL: 15 μL of 2 × Taq PCR MasterMix (containing Taq DNA polymerase, dNTPs, MgCl₂, reaction buffer, Beijing Adler Biotechnology Co., Ltd.), template DNA 3 μL, forward and reverse primers 1 μL each, ddH₂O 10 μL. The PCR amplification procedure was as follows: pre-denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and finally extension at 72°C for 10 min. After the PCR products were subjected to agarose gel electrophoresis, the samples with accurate and clear target bands were sent to Tianyi Huiyuan Biotechnology Co., Ltd. for purification, recovery, and sequence determination. The mitochondrial DNA Cyt b fragment was measured in both directions, and the D-Loop fragment was only sequenced in the reverse direction due to the presence of repeated fragments in the forward direction.

There are no published microsatellite [also known as simple sequence repeats (SSR)] primers for C. brachygnathus, but many microsatellite primers have been published for Coilia nasus. Because C. brachygnathus and C. nasus are closely related species, a total of 50 pairs of microsatellite primers of C. nasus were selected from references for primer screening with eight DNA templates of C. brachygnathus (Du et al., 2019; Yu et al., 2019). Finally, eight pairs of primers with higher polymorphism were screened out by the capillary electrophoresis method. The specific sequences, repeat motif, and optimum annealing temperature of each primer are listed in Supplementary Table 1. Then, the 5′ ends of the eight pairs of forward primers were labeled with fluorescent groups (FAM). The primer screening and fluorescent labeling of the primers were entrusted to Tianyi Huiyuan Biotechnology Co., Ltd. The total volume for the PCR was 10 μL: 5 μL of 2 × Taq PCR MasterMix (containing Taq DNA polymerase, dNTPs, MgCl₂, reaction buffer, Beijing Adler Biotechnology Co., Ltd.), template DNA 1 μL, forward and reverse primers 0.5 μL each, ddH₂O 3 μL. The PCR amplification procedure was as follows: pre-denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at suitable temperature for 40 s, extension at 72°C for 1 min, and finally extension at 72°C for 10 min. The amplified products were sent to Tianyi Huiyuan Biotechnology Co., Ltd. for microsatellite genotyping.

MEGA7 was used to align and rectify the nucleotide sequences by referring to the sequencing map (Kumar et al., 2016). DnaSP v5.10 was used to calculate the numbers of variable sites and haplotypes, haplotype diversity (H_d), nucleotide diversity (P_i), and gene flow (N_m) between groups (Librado and Rozas, 2009). T-test was used to test whether the genetic diversity parameters or gene flow of different groups are significantly different. Arlequin version 3.0 was used to perform analysis of molecular variance (AMOVA) and to compute pairwise genetic differentiation index F_ST values (Excoffier et al., 2005). MEGA7 was used to construct genetic distance trees by the neighbor-joining method (Kumar et al., 2016). Network 4.6 was used to construct haplotype network using the median-joining algorithm (Bandelt et al., 1999).

Genotypes were checked for large allele dropout, null alleles, and scoring errors due to stuttering by Micro-checker v2.2.1 (Van Oosterhout et al., 2004). Tests for deviations from Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD) across all pairs of loci were checked by GENEPOP v4.7.0 (Rousset, 2008) using exact tests with a Markov chain algorithm (P-values were estimated from 10,000 dememorizations, 100 batches, and 5,000 iterations per batch) (Guo and Thompson, 1992). Significance levels were adjusted for multiple comparisons using the sequential Bonferroni correction (Rice, 1989).

Number of alleles (A), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC) per locus were calculated by Cervus v3.0 (Kalinowski et al., 2007). Standardized allelic richness (Ar) was calculated using Fstat v2.9.3.2 (Goudet, 2001). T-test was used to test whether the genetic diversity parameters or gene flow of different groups are significantly different. Arlequin v3.0 was used to perform AMOVA and to compute pairwise genetic differentiation index F_ST and gene flow index N_m (Excoffier et al., 2005). In addition, genetic differentiation among populations was depicted by two-dimensional plots from a principal components analysis (PCA) of the allele frequencies matrix in GENALEX 6.4 (Peakall and Smouse, 2006).

The number of genetically differentiated clusters (K) was inferred using Bayesian assignment analysis by STRUCTURE v2.3.4 (Pritchard et al., 2000). Each assumed K (1–12) were performed 10 replications under an admixture model and correlated allele frequencies within populations (400,000 iterations with 100,000 burn-in periods) (Falush et al., 2003). The optimal K was selected according to the mean log probability LnP (K) and the ΔK value for each K (Evanno et al., 2005) using Structure Harvester (Earl and vonHoldt, 2012).¹

Identifying the invasion source and pathway of C. brachygnathus in the Three Gorges Reservoir was the top priority in this study, because the acquisition of this information is important for the prevention and control of C. brachygnathus in the Three Gorges Reservoir (Ficetola et al., 2008). Through this study, we speculated that the main invasion source of C. brachygnathus in the Three Gorges Reservoir was the middle Yangtze River instead of the lower Yangtze River. There were several results supporting this speculation. Firstly, the genetic differentiation, genetic distance between the Three Gorges Reservoir and the middle Yangtze River were smaller than between the Three Gorges Reservoir and the lower Yangtze River, and the gene flow result was opposite. Secondly, network of haplotypes indicated that there were shared haplotypes between the Three Gorges Reservoir populations and the middle Yangtze River populations, but no shared haplotypes between the Three Gorges Reservoir populations and the lower Yangtze River populations; the STRUCTURE analysis showed that the Three Gorges Reservoir populations only have some of the same genetic cluster components as the middle Yangtze River populations; PCA showed that the Three Gorges Reservoir populations only have some overlapping areas with the middle Yangtze River populations. Yang (2019) also found that the genetic distance of C. brachygnathus between the Three Gorges Reservoir populations and Dongtinghu Lake population was small. The reason for the middle Yangtze River population being the main invasion source, instead of the lower Yangtze River population, of C. brachygnathus in the Three Gorges Reservoir may be related to geographic distance and the dispersal ability of C. brachygnathus (Radinger and Wolter, 2014; Radinger et al., 2017). Due to limited diffusion capacity, C. brachygnathus in the middle reaches of the Yangtze River can spread to the Three Gorges Reservoir, while C. brachygnathus in the lower Yangtze River hardly spread to the Three Gorges Reservoir.

As for the invasion pathway, we speculated that the most probable pathway for C. brachygnathus to have invaded the Three Gorges Reservoir was individuals from Poyang Lake, Dongting Lake, Changhu Lake, and other middle reaches of the Yangtze River actively moving upstream to the Three Gorges Reservoir through the operation of ship locks. When the ship was transported from under the dam to above the dam by cascade ship lock, the fish were also transported up. Lin et al. (2013) combined fish catch surveys and hydroacoustic detection to explore the fish passing ability of the ship locks of the Gezhouba and Three Gorges Dam; in the fish catch surveys of the two locks, a total of 21 species were identified, and the fish catch at the Gezhouba ship lock contained C. brachygnathus. Consistent with this, Xiang et al. (2015) used sonar (DIDSON) to detect and record the number of fish crossing up and down the Gezhouba ship lock and found that fish were able to pass the ship lock under natural conditions. Therefore, it is reasonable that C. brachygnathus spread to the Three Gorges Reservoir through the ship locks.

For many invasive species, the level of genetic diversity of an invaded area is often lower than that in the area of origin, because the invaded population is often produced from a few individuals from the original area; this is known as the founder effect (Allendorf and Lundquist, 2003; Crawford and Whitney, 2010; Peischl and Excoffier, 2015). In the present study, whether considering mitochondrial genes or microsatellites, the genetic diversity of C. brachygnathus in the non-invaded habitats (TH, AHCH, PYH, DTH, CH) was significantly greater than that in the invaded habitats (SDP, TPX, XXH, XJ, WZ, FD, FL), indicating that the invaded populations experienced a founder effect. However, some studies have found that the genetic diversity of invasive species in the invaded region was not lower than that in the original area or was even higher than that in the area of origin (Frankham, 2005; Stepien et al., 2005). For example, Diez-del-Molino et al. (2013) found that Mosquitofish populations from the invaded Spanish streams had similar levels of genetic diversity as in the native American samples, suggesting these invasive populations did not appear to have undergone substantial loss of genetic diversity during the invasion process. The occurrence of this anomaly might be attributed to a single large number of individual invasions or to multiple introductions (Roman and Darling, 2007; Dlugosch and Parker, 2008). A single invasion of a large number of individuals makes the invading individuals include almost all the genetic diversity of the original population, and multiple introductions comprise genetic diversity of multiple ranges. Hybridization between individuals from different sources will also increase the genetic diversity of the population (Ellstrand and Schierenbeck, 2006).

The above discussion suggests that among the successful invasion cases, there are two contrasting patterns of genetic diversity comparisons of native and invaded ranges for different invasion cases. Therefore, what is the relationship between genetic diversity and successful invasion? It is generally believed that a high level of genetic diversity is beneficial to the settlement and maintenance of alien species in the new environment (Kolbe et al., 2004; Crawford and Whitney, 2010; Kanarek and Webb, 2010). Low levels of genetic diversity may be accompanied by reduced heterozygosity and inbreeding depression, restricting the population’s evolutionary potential and ability to adapt to environmental changes and increasing the risk of population extinction (Altizer et al., 2003; Pinsky and Palumbi, 2014). Reed and Frankham (2003) conducted a meta-analysis of 34 research studies involving genetic diversity and population fitness and found that the level of population genetic diversity was significantly positively correlated with population fitness. However, in the present study, the genetic diversity of the invasive C. brachygnathus was relatively low due to founder effect. Why then has the C. brachygnathus invasion been so successful? We speculate that the successful invasion of C. brachygnathus could be related to its life history characteristics and its niche in the fish community of the Three Gorges Reservoir. C. brachygnathus has sufficient food, high fecundity, and strong ability to resist predation in the Three Gorges Reservoir, as introduced in the introduction, and these factors provide the species a competitive advantage over other fish. Therefore, the relationship between fish genetic diversity and successful invasion is not a simple causal relationship. The success of fish invasion is related not only to the genetic diversity but also to the life history characteristics of the invasive species and the environmental conditions.