Genetic Diversity and Phylogeographic Structure of Icefishes (Salangidae) from Chinese Freshwater Lakes

Icefishes belong to the Salangidae family (Zhang et al., 2007). They exhibit ecological traits of r-selection, including high fecundity, short generation cycles, early maturity, minimal parental care and strong colonization ability in unstable environments (Zhang et al., 2013). Globally, icefishes comprise approximately 20 species in six genera, primarily distributed in the coastal and inland waters of East Asian countries and regions (Saruwatari et al., 2002). In China, 15 icefish species in six genera have been classified based on morphological characteristics, with main distributions in Yangtze River system (Xie and Chen, 1999). For instance, due to its great commercial significance, Neosalanx taihuensis has been introduced into a large number of lacustrine and reservoir systems across over 20 Chinese provinces since the 1980s (Liu, 2001). This species can rapidly adapt to new conditions by evolving phenotypic or genotypic changes within a short number of generations. Such adaptation often occurs through natural selection acting on pre-existing genetic variation or new mutations, ultimately leading to population differentiation or the emergence of new species (Fang et al., 2022). 
       
Icefishes disperse via artificial introduction and natural swimming-based diffusion. The introduction and reproduction of Chinese icefishes in freshwater lakes primarily involve N. taihuensis, N. pseudotaihuensis and Protosalanx hyalocranius (Wang et al., 2002). Yunnan has records of successive introductions of N. taihuensis into Dianchi, Xingyun and Erhai lakes in the early 1980s (Chen et al., 1989; Gao et al., 1989; Wu et al., 1998). Zhou and Xie (1991) confirmed that icefishes introduced into Dianchi Lake were N. taihuensis and N. pseudotaihuensis. Genetic differentiation may exist among these geographical populations, including potential cryptic or disputed species. However, due to overfishing, environmental pollution and habitat destruction, icefish populations have continuously declined and their distribution ranges significantly reduced. Some species are gradually becoming endangered (Zhang, 2008) and exhibit low genetic diversity, which generally indicates poor environmental adaptability (Rossi et al., 2021). When environmental pressures (such as habitat destruction) reduce population size, genetic drift intensifies random fluctuations in allele frequencies, thereby rapidly decreasing genetic diversity (Frankham, 1995). Therefore, a comprehensive understanding of the genetic diversity and phylogeographic structure of these populations is crucial for enhancing the protection and rational utilization of icefish resources.
       
Mitochondrial DNA (mtDNA) is distinguished by its simple structure, maternal inheritance and rapid mutation rate (Lai et al., 2015) and has been widely used in research on vertebrates genetic diversity, population structure and phylogeny (Naseer et al., 2018; Utami et al., 2024). Among cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) are ideal molecular markers for species and genus level phylogenetic studies (Abbas et al., 2022; Sari et al., 2015). Currently, researchers have used mitochondrial and microsatellite DNA markers to investigate the genetic diversity (Fang et al., 2022; Xing et al., 2022; Zheng et al., 2024) and systematic evolution (Liu et al., 2018) of icefishes. However, existing studies mainly focus on the genetic variation patterns in Protosalanx chinensis, N. taihuensis, Salanx ariakensis and N. jordani using Cytb sequences. To date, knowledge regarding the genetic diversity and phylogeographic structure of icefishes in Chinese freshwater lakes based on COI and Cytb genes remains scarce.
       
In this study, we utilized COI and Cytb sequences to analyze the genetic diversity and phylogeographic structure of seven icefish populations. The primary objectives were (1) to investigate genetic diversity of seven icefishes populations and formulate rational conservation strategies; (2) to determine whether significant phylogeographic structure exist within these populations; (3) to identify whether there are other icefish species besides N. taihuensis in these populations. These findings of this research can serve as a reference for the protection and utilization of icefish resources.

Sample collection and DNA extraction
 
In total, 144 and 140 icefish samples were collected from Yunnan seven freshwater lakes. After collection, these samples were preserved in Zoology Laboratory of the College of Life Sciences of Yunnan Normal University for COI and Cytb gene analysis, respectively (Table 1). The Biomedical Research Ethics Committee of Yunnan Normal University approved all the experimental protocols applied in this study. We used the Hi-Pure Animal Genomic DNA Kit (TSINGKE TSP202-200 Trelief) following the kit’s protocol precisely to extract genomic DNA from the dorsal muscle. After the DNA concentration was detected, these samples were stored at -20°C for subsequent analysis. The entire research period was from January 2023 to April 2024.


 
PCR amplification and sequencing
 
The COI fragment was amplified using primers Fish-F1 (5'- TCGACTAATCATAAAGATATCGGCAC-3') and Fish-R1 (5'-TAGACTTCTGGGTGGCCAAAGAATCA-3') (Ward et al., 2005). The Cytb gene primers were L14321 (5'-CC AGTGACTTGAAAAACCACCG-3') and H15634 (5'-CT TAGCTTTGGGAGTTAAGGGT-3') (Zhang et al., 2007). PCR amplifications of two genes took place within a 50 μL reaction system. This reaction mixture included 2 μL of each 10 μmol/L primer, 2 μL of genomic DNA, 25 μL of 2 × San Taq PCR Mix and19 μL of ddH₂O. PCR cycling conditions of COI and Cytb genes were initial denaturation at 94°C for 5 min, followed by 35 cycles (COI) or 30 cycles (Cytb) of denaturation at 94°C for 30 s, annealing at 50°C for 35 s (COI) or 55°C for 50 s (Cytb) and extension at 72°C for 45 s (COI) or 90 s (Cytb), with a final extension at 72°C for 8 min (COI) or 10 min (Cytb) (Ward et al., 2005; Zhang et al., 2007). The successfully amplified PCR products were sent to Beijing Qingke Biotech (Kunming) Co., Ltd for bidirectional sequencing. Due to double peaks in some samples causing incomplete sequencing, the final usable sequences were 116 (COI) and 130 (Cytb).
 
Data analyses
 
The SeqMan program in the DNAstar v7.1 was employed to meticulously examine the newly obtained sequences. The Clustal W program integrated in MEGA7.0 to align for two genes sequences. The obtained sequences were used to calculate sequence similarity by the BLAST sequence analysis tool. We used the MEGA7.0 to calculate base composition and genetic distance of icefish populations based on Kimura 2-parameter method. The variation sites (S), parsimony informative sites (Np), number of haplotypes (Nh), haplotype diversity (Hd), nucleotide diversity (Pi) and gene flow (Nm) were calculated with DnaSP v5.10.
       
To determine phylogenetic relationship of seven icefish populations. A Neighbor-Joining (NJ) tree was constructed in MEGA7.0 using two obtained genes sequences, along with some Neosalanx species and outgroup (P. chinensis) sequences. Node supports in NJ tree analysis were evaluated using a bootstrap analysis with 1000 replications. The POPART software was used to construct the haplotype network to detect genetic relationships among haplotypes. Additionally, analysis of molecular variance (AMOVA) and a Mantel test were executed in Arlequin v3.5.2. To detect demographic history of seven icefish populations, the Neutrality tests (Tajima’s D, Fu’s Fs), the sum of squared deviation (SSD) and Raggedness index (r) were computed in Arlequin software.

MtDNA sequence characteristics and haplotypes
 
The 653 bp and 1205 bp fragment were successfully obtained for COI and Cytb gene, respectively. The proportion of A+T (45.2%, 48.6%) was lower than G+C (54.8%, 51.4%) for 116 COI and 130 Cytb sequences, which is consistent with previous research (Xiong et al., 2021; Zhong et al., 2016). Sequence similarity analysis revealed that COI sequence exhibited 100% identity with N. taihuensis  and N. tangkahkeii and 99% identity with Neosalanx sp. Cytb sequences showed 100% similarity with N. taihuensis  and N. tangkahkeii, confirming these icefish populations as N. taihuensis and N. tangkahkeii (Kang et al., 2015; Zhang et al., 2013). Amongst the 116 COI sequences, there were 8 variable sites, 4 parsimony informative sites and 8 haplotypes (Hap1C-Hap8C), with Hap1C as the core haplotype, accounting for 81.03% of all individuals. In the case of the 130 Cytb sequences, there were 17 variable sites, 12 parsimony informative sites and 13 haplotypes (Hap1B-Hap13B) (Table 1), with Hap2B as the core haplotype (67.69% frequency).
 
Genetic diversity and conservation of icefish resources
 
Genetic diversity underlies a species’ adaptability to environmental changes and supports evolutionary persistence (Roberts, 2002). Haplotype and nucleotide diversities were employed to evaluate genetic richness (Xing et al., 2022), with values >0.5 (Hd) and >0.005 (Pi) indicating high diversity (Grant and Bowen, 1998). This study found that the genetic diversity values (Hd=0.331±0.053, Pi=0.00059±0.00011) of the COI gene were lower than the average values (Hd=0.576±0.036, Pi=0.00112±0.00204) calculated by Zhang et al. (2012) and were consistent with the genetic diversity findings reported by Fang et al. (2022). However, the genetic diversity (Hd=0.525±0.050, Pi=0.00111±0.00018) of seven icefish populations based on the Cytb gene were greater than 0.5 and but less than 0.005. These results suggest a relatively low mt DNA diversity level, which may be attributed to long term overfishing, water quality degradation, competition with other fish species and artificial control (Avise, 2004). This phenomenon could also be due to the genetic bottleneck often occurring during the introduction process and the potential founder effects resulting from the introduction of a small number of individuals (Fang et al., 2022; Zhu et al., 2023). For example, Fang et al. (2022) analyzed the genetic diversity of N. taihuensis from eleven Chinese river basins and found high haplotype diversity but low nucleotide diversity, displaying that N. taihuensis may have undergone rapid population expansion following bottleneck effects. However, this study only used mitochondrial markers, which may not fully represent genetic variation. Nuclear markers (such as microsatellites and SNPs) are necessary for future validation, as these molecular markers have been widely used in genetic diversity research and phylogenetic studies of various animals (Veeramani et al., 2023; Pan et al., 2023). Hence, it is necessary to strengthen resource conservation measures to prevent the loss of genetic variation. At the population level, the YZH population’s higher diversity (Table 1) may reflect larger sample size rather than true genetic variation, highlighting the need for standardized sampling across all populations.
       
A “unified management unit” refers to a coordinated and integrated entity that oversees various aspects of fishing activities, like resource monitoring and catch limits, to ensure sustainable yields in fisheries (Badr et al., 2014). Studies on genetic diversity and genetic structure of fish populations can provide crucial references for establishing fishery resource management measures and species conservation strategies (Avise, 2004). This study found relatively low genetic diversity and limited variation among seven icefish populations, indicating poor genetic resources in these lakes. Therefore, these populations should be conserved as a unified management and protection unit. Currently, effective control measures should be implemented for these icefish populations. Such as, avoiding overfishing, protecting habitats, reducing environmental pollution and improving the ecological environment of icefish in the process of management and conservation. As icefishes are one year lived species with a short life cycle and a high reproduction rate, the breeding and release of icefish populations should be carried out scientifically and regularly. Moreover, monitoring and genetic assessment of migratory and wild populations should be strengthened to enhance genetic diversity of icefishes in Yunnan lakes.

Phylogeographic structure of populations
 
Calculating genetic distances in fish research distinguishes species precisely, unravels evolutionary histories and identifies cryptic species, aiding taxonomy and conservation (Hebert et al., 2003). The COI sequences of N. tangkahkeii, Neosalanx sp., N. taihuensis, P. chinensis (Table S1) and our samples sequences were combined to calculate genetic distances (Table 2), indicating that the genetic distance between our measured samples and Neosalanx sp. was the smallest (0.1%). While the genetic distance between our measured samples and N. tangkahkeii (0.2%) and N. taihuensis (0.2%) were significantly lower than 2% (Hebert et al., 2003), indicating an intraspecific level. The COI tree indicated that seven populations samples, N. tangkahkeii, Neosalanx sp. and N. taihuensis were gathered into a monophyletic clade with 100% bootstrap support rate (Fig 1a). Notably, icefish samples from each lake did not cluster independently, but were interspersed with samples from other lakes. The Cytb tree results was similar to that of COI tree (Fig 1b). As of 1993, N. taihuensis was transplanted into Yunnan eleven lakes (Zhuang et al., 1996). Therefore, it is speculated that N. taihuensis and N. tangkahkeii are present in the seven lakes of Yunnan. However, Zhong et al. (2016) reported that sequence similarity between N. tangkahkeii and N. taihuensis was 99.9% and suggested that they belong to the same species. Thus, precise species distinction not only prevents over exploitation of specific populations and maintains ecosystem balance in fisheries, but also aids in understanding speciation mechanisms and adaptive evolution. This provides a scientific basis for addressing challenges such as habitat fragmentation and climate change (Murat, 2025).






       
The NJ tree and haplotype network (Fig 2) demonstrated that samples or haplotypes of each icefish population from seven lakes did not cluster separately and haplotypes were shared among individuals from different populations. The AMOVA analysis indicated that the genetic variation occurred predominantly within population (89.97% for COI, 76.61% for Cytb), despite the populations being geographically separated. A Mantel tests displayed no significant associations (R=-0.1717, P=0.7796 for COI; R=-0.2374, P=0.8017 for Cytb) between genetic and geographic distances (Table S2), which is similar to genetic structure studies on P. chinensis (Zheng et al., 2024). This phenomenon may be explained by extensive gene flow (2.35 for COI, 0.90 for Cytb). Collectively, these results demonstrated no significant phylogeographic structure among these icefish populations. As economically valuable small fish, icefishes spread via cruise ships and human activities, promoting gene exchange. However, the absence of phylogeographic structure has significant impacts on fisheries management. In this study, treating all populations as unified management unit has protected icefish, but may have overlooked local adaptive traits. This is because unmonitored intensive fishing could damage genetically distinct subpopulations and climate change may also disrupt gene flow, thus urgently requiring the formulation of dynamic management plans (Murat, 2025).




 
Demographic history
 
Neutrality tests (Fu, 1997; Ramos-Onsins and Rozas, 2002) and mismatch distribution analyses (Barbosa et al., 2013) can be used to predict historical demographic expansions. As expected, all seven populations dataset indicated population expansion (Table 1; Fig 3). The haplotype network presented a star-like phylogeny. This is a characteristic trait typically associated with a population that has experienced a recent expansion (Avise, 2004). Additionally, the statistical tests of SSD and Raggedness were not significant, suggesting no significant deviation from the population expansion model. These results are in accordance with earlier investigations (Fang et al., 2022).

Our results revealed relatively low mtDNA diversity across seven Yunnan icefish populations. As expected, no significant phylogeographic structure was detected among these populations and all seven populations datasets presented indications of population expansion. Besides N. taihuensis, N. tangkahkeii may also inhabit these seven freshwater lakes. These findings of this study indicated that different populations ought to be regarded as an integrated management unit to achieve effective conservation and management goals. Future research will incorporate nuclear molecular markers and genome-wide analysis to further characterize genetic diversity in Yunnan icefish populations.

This study was funded by the National Natural Science Foundation of China (32060115, 31560589, 31260507), the Basic Research Special General Project of Yunnan Provincial Department of Science and Technology (202401AT070101),  the Scientific Research Fund of Yunnan Provincial Education Department (2024J0140) and College Students Innovation Training Program Project of Yunnan Province (s202410681070).
 
Disclaimers
 
The opinions and conclusions in the article only represent those of the authors and do not necessarily represent their affiliated institutions.
 
Informed consent
 
The Biomedical Research Ethics Committee of Yunnan Normal University had approved experimental procedure that used animals in this research.

All authors state that there are no conflicts of interest.