Genetic diversity and differentiation of Daphnia galeata in the middle and lower reaches of the Yangtze River, China

Abstract Mitochondrial 16S rDNA and CO I gene were used as molecular markers for the analysis of the genetic diversity and differentiation of Daphnia galeata populations in nine water bodies in the middle and lower reaches of the Yangtze River. In the combined 16S rDNA and CO I gene sequences, 54 variable sites and 44 haplotypes were observed among 219 individuals belonging to nine D. galeata populations. Average haplotype diversity and nucleotide diversity were, respectively, 0.72% and 0.56%. The F‐statistics (F ST) value of the D. galeata populations was 0.149. According to the results of the neutral test, D. galeata in the middle and lower reaches of the Yangtze River had experienced a bottleneck effect in the history. Molecular variance analysis indicated that the genetic differentiation of the D. galeata populations mainly occurred within populations (85.09%). Greater genetic differentiations of D. galeata among individuals within populations appeared in the populations from the Huaihe River basin, whereas smaller genetic differentiations occurred in the populations from the middle reaches of the Yangtze River. Strong gene flows were all observed between Group I (four populations from the middle reaches of the Yangtze River) and Group ΙΙ (three populations from the middle and lower reaches of the Yangtze River), and Group ΙΙΙ (two populations from the Huaihe River basin). The effective migration rates (M) were 851.49 from Group I to Group ΙΙ and 685.96 from Group I to Group ΙΙΙ, respectively. However, no significant relationship was observed between the genetic differentiation and geographical distance of the nine populations (r = .137, p > .05). Results suggested that the genetic differentiation of D. galeata in the water bodies in the middle and lower reaches of the Yangtze River resulted mainly from geographical isolation.

the genetic structure of eight populations of the D. longispina complex in Eastern China and found that only D. galeata appeared in the investigated lakes. In addition, D. galeata was also detected as the main species in eutrophic lakes located in central and eastern China Xu et al., 2018). Usually, Daphnia species have high dispersal capabilities (Louette & De Meester, 2005). How the adaptability of Daphnia to newly ecological environments or how to evolve in a watershed system is not well known.
The genetic diversity and phylogenetic evolution of Daphnia species have been extensively studied on the basis of molecular markers. Based on the haplotype diversity (Hd) and nucleotide diversity (π) of mitochondrial gene sequences, Grant and Bowen (1998) classified the genetic diversity of marine organisms into four types: (a) low Hd (<0.5) and low π (<0.5%), suggesting recent population bottleneck or founder event; (b) high Hd (>0.5) and low π (<0.5%), indicating an expansion after a period of low effective population size and enhanced retention of new mutations; (c) low Hd (<0.5) and high π (>0.5%), suggesting the secondary contact between isolated populations or a strong bottleneck in a formerly large and stable population; (d) high Hd (>0.5) and high π (>0.5%), denoting the high level of divergence between haplotypes. Hebert, Witt, and Adamowicz (2003) observed that long-time geographical segregation resulted in genetic differences among Daphnia ambigua populations in five different geographical regions. Xu et al. (2018) found that the genetic distance differences between 10 Daphnia distributed in different water bodies of the Tibetan Plateau were 9.25%-30.71%. A high genetic differentiation was observed among Daphnia magna species in Europe, North America, and Japan (De Gelas & De Meester, 2005), suggesting an intercontinental diffusion of the species. Compared with the D. magna populations living in the medium-sized and stable ponds of southern or central Europe, those located in small rock pools of northern Europe have significantly low genetic diversity and high genetic differentiation (Walser & Haag, 2012). By examining 49 screened populations of D. longispina and 77 populations of D. magna, Haag, Riek, Hottinger, Pajunen, and Ebert (2005) found that local genetic diversity increased with population age, whereas pairwise differentiation among populations decreased. Straughan & Lehman (2000) found that the genetic differences of Daphnia pulex complex among different lakes were affected by river basin location, regional physical geography, bird migratory flyways, and lake trophic status. Meanwhile, D. longispina complex, D. galeata, and Daphnia dentifera living in the New North District had introgression in Quaternary glacial activity (Ishida et al., 2011). Daphnia galeata was mainly distributed in eutrophic lakes in low-altitude areas in central and eastern China, whereas D. dentifera was distributed in high-altitude oligotrophic lakes in the Tibetan Plateau in western China .
The Yangtze River Basin is a concentrated area of freshwater lakes in China, where many lakes have become eutrophic (Deng et al., 2008;Huang, Gao, & Zhang, 2016;Qin, 2002;Wu, Qin, & Yu, 2016). Two major river systems, the Yangtze River and the Huaihe River, run through this basin. Historically, the Huaihe River drained directly into the Yellow Sea, but it is now connected to the lower reaches of the Yangtze River after many floods (Wu, Wang, et al., 2019a). Along the Yangtze River Basin, many tributaries of the river and lakes are distributed, which is an ideal habitat for studying the genetic differentiation of Daphnia.  observed the genetic differentiation among D. pulex populations distributed in 10 water regions in the middle and lower reaches of the Yangtze River. Wang (2016) also found genetic differences among Daphnia similoides sinensis from the hatching of resting eggs in different sedimentary layers of Lake Chaohu. Wu, Wang, et al. (2019a) found that D. similoides sinensis from seven water bodies located in the middle and lower reaches of the Yangtze River had evolved into two clades, namely the Yangtze River clade and the Huaihe River clade. In Lake Junshan, Wu, Zhang, et al. (2019b) observed that there were obviously genetic differences of D. similoides sinensis before and after the construction of artificial dam. Similarly, some genetic differentiation of D. galeata populations from lakes or reservoirs located in the upper or lower reaches of the Yangtze River was found, and these differentiations were not related to geographical distance (Wei et al., 2015;Xie et al., 2015). Although there were some investigations on the genetic differentiation of Daphnia in the Yangtze River, it was fragmented and unsystematic for D. galeata in the area.
In this study, we selected 16S rDNA and CO I gene as molecular markers to investigate the genetic diversity and differentiation of D. galeata in nine water bodies in the middle and lower reaches of the Yangtze River and explore the relationship between genetic differentiation and geographical distribution pattern. This study will increase understanding of the genetic structure and global phylogenetic evolution of D. galeata.

| Animal culture
Daphnia galeata (Figure 1) was collected from two water bodies of Huaihe River basin (Lake Nanhu, Huaihe River section in Bengbu city) and seven lakes in the middle and lower reaches of the Yangtze River (Lake Junshan, Lake Longgan, Lake Pohu, Lake Wuchang, Lake Chaohu, Lake Taihu, and Lake Dianshan) (Table 1, Figure 2). The identification of D. galeata was according to the methods of Jiang and Du (1979) and Benzie (2005). The collected D. galeata individuals were monoclonally cultured in an intelligent lighting incubator (Ningbo Saifu), with the illumination of 12 hr light: 12 hr dark at (25 ± 1)°C, and 4-6 mature females per individual were selected for DNA extraction. The culture medium was changed every day, and D. galeata were fed with Scenedesmus obliquus of 2 × 10 5 cells/ml. The culture medium was aerated tap water over 48 hr, filtered by boiling and cooling for reserve.

| DNA extraction and PCR amplification
The genomic DNA of D. galeata was extracted by using TIANamp Micro DNA Kit (Tiangen). Each D. galeata body was crushed with a sterile 10 μl tip before extraction because the chitin carapace of D. galeata hindered the digestion of internal organs by proteinase K. The concentration of DNA extraction was measured by a spectrophotometer (Biofuture) and then stored at −20°C for subsequent analyses.

| Data analyses
We compared the alignments of the D. galeata 16S rDNA and CO I gene sequences in the NCBI database to ensure the reliability of our sequences. Before the combined analysis of 16S rDNA and CO I gene sequences, the homogeneity of the two gene sequences was tested by using PAUP*v4.0, and the results showed that the topologies of the two data sets were consistent (p = .14). Therefore, the two gene sequences can be combined.
The homologous alignment of 16S rDNA and CO I gene sequences was performed by using Mega 7.0, and the missing se- Haploviewer (Salzburger, Ewing, & Haeseler, 2011). The directional gene flow was analyzed by Migrate-n version 3.6 software using the strategy of Bayesian inference and constant mutation rate (Beerli, 2006;Beerli & Palczewski, 2010). The mutation-scaled population sizes Ɵ and immigration rates M were calculated. The calculation formula of gene flow (Nm) is X × Nm = Ɵ × M, where X is the inheritance parameter and depends on the molecular marker, which is usually 1 for mt DNA data (Beerli, 2006;Beerli & Palczewski, 2010). A circle map of directional gene flow was drawn through the online version of the software CIRCOS (Krzywinski et al., 2009).   (Hd = 0.13). In the CO I gene sequences, a high haplotype diversity (0.33-0.81) was found, and the highest and lowest haplotype diversity appeared in the DS population (Hd = 0.81) and JS population (Hd = 0.33), respectively. In the 16S rDNA gene sequences, the highest nucleotide diversity appeared in AH population (π = 0.50%) whereas the lowest occurred in JS and WC populations (π = 0.02%). The overall nucleotide diversity π of nine D. galeata populations was 0.28%. In the CO I gene sequences, the highest nucleotide diversity appeared in the AH population (π = 1.57%)

| Genetic diversity of D. galeata populations from the middle and lower reaches of the Yangtze River
whereas the lowest occurred in the JS population (π = 0.08%). The overall nucleotide diversity π of nine D. galeata populations was 0.91% (Table 2).
Under the combined sequences of the 16S rDNA and CO I genes,

| Genetic differentiation of D. galeata population in the middle and lower reaches of the Yangtze River
The genetic distances of D. galeata populations from nine water bodies in the middle and lower reaches of the Yangtze River ranged from 0.001 to 0.010, and the minimum value appeared in the JS, LG, and WC populations. Meanwhile, the maximum value was obtained in the AH population. Some differences were observed between AH population and other populations, ranging from 0.009 to 0.010 (Table 3).
In terms of geographical position, Lake Junshan had close distance to Lake Longgan and Wuchang. However, the F-statistics (F ST ) values of the D. galeata populations among the JS, LG, and WC populations were all relatively high. Lake Chaohu was far away from Lake TA B L E 3 Genetic distances between or within populations of Daphnia galeata from water bodies in the middle and lower reaches of the Yangtze River based on the combined sequences of 16S rDNA and CO I genes

| D ISCUSS I ON
DNA sequencing has extensively been used as molecular tool on modern taxonomic and biogeographical research (Ratnasingham & Hebert, 2007). Usually, mitochondrial DNA (mtDNA) is relatively conservative, having a strict maternal inheritance. Both 16S rDNA and CO I genes are two popular markers in identifying the genetic differentiation and phylogeny of Daphnia species Ma, Petrusek, Wolinska, Hu, & Yin, 2019;Xu et al., 2018Xu et al., , 2014. In the present study, the A + T contents were all higher than the C + G contents in the 16S rDNA and CO I gene sequences of D. galeata, which was consistent with other Daphnia species (Hebert et al., 2003;Wu, Wang, et al., 2019a;Xu et al., 2018). Moreover, our results suggested that the combined analysis of the 16S rDNA and  individuals within the same population. The higher the genetic diversity, the stronger the ability of the individuals in the population adapted to environmental changes (Haag et al., 2005). According to Grant and Bowen (1998), the genetic diversity of marine organisms was classified by haplotype diversity (Hd) and nucleotide diversity (π) of mitochondrial gene sequences. In this study, nine D. galeata populations in the middle and lower reaches of the Yangtze River showed high genetic diversity (Hd = 0.74, π = 0.57%). However, the genetic diversity among populations was significantly different. In Lake Junshan, strong predation pressure of fish evidently inhibited the population density of D. galeata (Zhao, Zhang, Peng, Zhang, & Deng, 2018). Owing to its short growing season, the genetic diversity of the D. galeata population encountered strong clonal erosion, resulting in low haplotype diversity (Huang, Xu, Xu, & Han, 2017).
This phenomenon might be an important reason for low haplotype and nucleotide diversity (Hd = 0.34 and π = 0.05%) of JS population. Previous reports showed that floods had frequently occurred in the middle and lower reaches of the Yangtze River from 1921 to 2000 (Shi, Jiang, Su, Chen, & Qin, 2004;Xu, Yu, & Ma, 2005). The (Hd = 0.65 and π = 0.70%). Lake Nanhu, which is a small closed lake formed by coal mining collapse, is located in the warm temperate monsoon climate zone. With the continuous deterioration of the lake environment, Lake Nanhu had gradually developed at a eutrophic level (Deng et al., 2010). The polytropic environmental conditions could provide a change for the coexistence or seasonal succession of different D. galeata genetic populations and then compensate for the formation of D. galeata with high genetic diversity (Xie et al., 2015). Lake Nanhu is also a habitat for some kinds of waterfowls and an important transfer point of some migratory birds in the middle and lower reaches of the Yangtze River (Wang, Jian, Zhang, & Zhou, 2016). Previous studies had indicated that the resting eggs of zooplankton could be attached to the feathers of birds for long-distance transmission (Figuerola, Green, & Michot, 2005;Proctor, 1964), which played an important role in the gene flow among Daphnia populations. Therefore, diverse environmental factors and bird migration might be important reasons for the high genetic diversity of the HN population of D. galeata. Posada and Crandall (2001) believed that ancient haplotypes located at the center of species evolution had the characteristics of high frequency and wide distribution and then spread to other haplotypes. In this study, Hap3 was located not only in the center of haplotype network but also in all nine D. galeata populations. Therefore, Hap3 might be regarded as the ancient haplotype in D. galeata populations in the middle and lower reaches of the Yangtze River.
Notably, the proportion of Hap3 in D. galeata populations in the middle reaches of the Yangtze River (LG, WC, PH, and JS) was evidently higher than those found in the lower reaches of the Yangtze River (DS, TH, and CH) and the Huaihe River Basin (HN and AH).
This finding might be caused by the spread of D. galeata populations. Bohonak and Jenkins (2003) found that species diffusion could promote biological evolution. Zooplankton (e.g., Daphnia) and their resting eggs could easily expand to other water bodies through passive diffusions, such as bird migration (Figuerola et al., 2005;Proctor, 1964), fish migration (Mellors, 1975), water diffusion (Michels et al., 2010), and wind transmission (Bilton, Freeland, & Okamura, 2001), In the present study, the direction of D. galeata gene flow is mainly from the middle reaches to the lower reaches of the Yangtze River. This pattern of gene flow was inconsistent with classic pattern which was from an older and more genetically variable population to a younger and less genetically variable population. In several lakes in southern and north-west Germany, Mantel tests showed a highly significant decrease in Nm (gene flow) with distance for populations of Daphnia hyalina (Sabine, 1997). In China, the lower reaches of the Yangtze River had quicker economic development than the middle reaches in past decades. The environmental pollution (especially eutrophication) of the lakes (Lake Chaohu, Lake Taihu, Lake Dingshan) in the lower reaches of the Yangtze River was more serious than those of the lakes (Lake Longgan, Lake Pohu, Lake Wuchang, Lake Junshan) in the middle reaches (Table 1). Higher nutrient concentrations (particularly nitrogen and phosphorous) accelerated the growth and development of phytoplankton, provided more food resources for cladocerans (including Daphnia), and then expanded Daphnia populations (Deng et al., 2008;Rellstab, Keller, Girardclos, Anselmetti, & Spaaka, 2011). In Swiss lakes, Spaak et al. (2012) thought that environmental changes (i.e., eutrophication) and local Theoretically, the frequency of individual communication between populations with close geographical distances should be larger than that between populations with far geographical distances. Population segregation or population genetic differences were positively correlated with geographical distances (Hutchinson, 1967 (Louette & De Meester, 2005), in contrast to its high dispersal capacity; however, D. galeata has been found to exhibit strong population genetic differentiation, even over small geographical scales (De Meester, 1996). In the middle and lower reaches of the Yangtze River of China, the linkage between the Huaihe River and Yangtze River was changed in 1953 after the construction of Sanhe sluice (Wu, Wang, et al., 2019a), and the interflow of D. galeata between the two rivers was also limited. Similarly, Lake Junshan is a typical isolated lake from Lake Poyang, and the connection of Lake Junshan with Lake Poyang and the Yangtze River were cut off after the construction of the lake embankment . This phenomenon hindered the gene flow of D. galeata between populations and led to evident genetic differentiations of D. galeata among Lake Junshan and its neighboring lakes. In this study, weak gene exchanges of D. galeata between the CH population and the populations in the middle reaches of the Yangtze River were found, whereas strong gene exchanges were observed in the populations in the lower reaches of the Yangtze River. Lake Chaohu is linked to the Yangtze River through the Yuxi River (Wang & Dou, 1998) and could strengthen the gene exchanges between the CH population and the populations in the lower reaches of the Yangtze River. Furthermore, Lake Chaohu and Lake Taihu had the developed navigation function. The water system of Lake Dianshan had also a certain connection with Lake Taihu (Wang & Dou, 1998). Historically, frequent floods had occurred in the middle and lower reaches of the Yangtze River (Shi et al., 2004;Xu et al., 2005). Similarly, the genetic differentiation of other Daphnia species from water bodies in the middle and lower reaches of the Yangtze River had been observed at different levels Wei et al., 2015;Wu, Wang, et al., 2019a). Wei et al. (2015) found that three genetically differentiated D. galeata subgroups from eight lakes in the lower reaches of the Yangtze River were observed and did not cluster by their geographical origin in overall.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The sequence data of Daphnia galeata in this study have been de-