Genetic diversity and phylogeography of Daphnia similoides sinensis located in the middle and lower reaches of the Yangtze River

Abstract Geographical patterns, climate, and environmental change have important influences on the distribution and spread of aquatic organisms. However, the relationships between the geographical pattern and phylogenetics of Daphnia as well as environmental change are not well known. The genetic diversity and phylogeography of seven D. similoides sinensis populations located in the middle and lower reaches of the Yangtze River were investigated based on the combination of mitochondrial (COI gene) and nuclear (14 microsatellite primers) markers. Based on the mitochondrial gene markers, D. similoides sinensis from the middle and lower reaches of the Yangtze River had one ancestral haplotype and two evolutionary clades. In addition, D. similoides sinensis population deviated from neutral evolution, showing signs of a bottleneck effect followed by population expansion. Based on the microsatellite markers, the seven D. similoides sinensis populations formed three main groups. The dendrogram (NJ/ME) showed that D. similoides sinensis based on the mitochondrial genes marker were obviously clustered two main clades, whereas there were three clades based on the microsatellite markers. Our results suggested that the habitat fragmentation due to the barrier of the dams and sluices promoted the genetic differentiation and phylogeography of D. similoides sinensis populations in the middle and lower reaches of the Yangtze River.


| INTRODUC TI ON
Geographical patterns, climate, and environmental changes have important influences on the genetic composition, population distribution, and species diversity of aquatic organisms (Hewitt, 2000;Petit et al., 2003). Avise et al. (1987) presented the concept of intraspecific phylogeography, whose basic principle was to study the relationship between gene genealogy and geography of organisms. The genealogical analysis and temporal-spatial distribution of haplotypes could be used to estimate the historical process of species differentiation between closely related species or at the intraspecific level (Avise, 1998;Zuykova, Bochkarev, & Sheveleva, 2016).
The phylogeography of organisms can be effectively revealed using multiple molecular markers. By using the mitochondrial COI and NDI genes, Ober, Matthews, Ferrieri, and Kuhn (2011) found that most mountain ranges resulted in the population differentiation of Scaphinotus petersi distributed on Sky Islands in southeastern Arizona during the postglacial maximum times. Based on the 16S rDNA, COI gene, and 18S rDNA molecular markers, Wang et al. (2016) concluded that the phylogenetics of the cladoceran Daphnia pulex located in ten habitats of the middle and lower reaches of the Yangtze River was related to its geographical location. In the nuclear genome, microsatellite markers have widely been applied to phylogeography because of their high polymorphism, stability, codominance, and Mendelian inheritance (Lane, Symonds, & Ritchie, 2016;Mobley, Small, Jue, & Jones, 2010).
Zooplankters are an important part of aquatic food chains and have important ecological roles in aquatic ecosystems. Daphnia is a common crustacean zooplankton, having the characteristics of wide distribution, rapid reproduction, and sensitivity to environmental changes (Su, 2013). So, Daphnia is often regarded as a model organism for the study of bio-toxicology, genetics, and ecology (Hebert, 1978;Lampert, 2011). Moreover, Daphnia has a weak swimming ability because of a small body size (Rand, 1996). D. similoides sinensis is distributed in eutrophic ponds and lakes in Southern Asia, from Pakistan to Indonesia, and China (Benzie, 2005). D. similoides sinensis perform cyclic parthenogenesis under good conditions, whereas they convert to sexual reproduction and produce resting eggs when environmental conditions worsen, such as low temperature, large predation pressure, or high population density (Figure 1). This species was previously recorded as D. similis or D. carinata in China (Gu, Xu, Lin, Henri, & Han, 2013;Jiang & Du, 1979;Xu et al., 2014). D. similoides sinensis was observed in some lakes located in the middle and lower reaches of the Yangtze River, China (Chen, Chen, Li, & Zhao, 2009;Ma et al., 2016), which coexisted with Daphnia pulex and Daphnia galeata (Deng et al., 2008).
The Huai River historically drained directly into the Yellow Sea, but it is now connected to the lower reaches of the Yangtze River after many floods. Along the middle and lower reaches of the Yangtze River and Huai River of China, many tributaries of the river and lakes are distributed ( Figure 2). There were nineteen floods in the middle and lower reaches of the Yangtze River from 1921 to 2000 (Shi, Jiang, Su, Chen, & Qin, 2004). Many dam and sluice projects in the region have been built since 1950s in order to store water or prevent the flooding, and some lakes have changed from a natural type into a reservoir type (Wang & Dou, 1998). In Lake Chaohu, the lake was isolated from the Yangtze River due to the construction of Chaohu dam and Yuxi dam in the 1950s. Similarly, the connection of Lake Junshan with Lake Poyang and the Yangtze River was cut off after the construction of the lake embankment in 1958 (Wang & Dou, 1998). In Wuhan city, Lake Nanhu had become a closed lake as a result of the development of the city. The building of dams and sluices can form a barrier for the migration and communication of aquatic organisms (Naiman, Melillo, Lock, Ford, & Reice, 1987;Yi, Yang, & Zhang, 2010), resulting in changes in species diversity or genetic diversity. About 28 species of fish have disappeared since 1950 in Lake Zhangdu due to the construction of artificial barriers between the rivers and lakes (Wang, Hu, & Wang, 2005). In the Three Gorges reservoir area of the Yangtze River, seven Leiocassis longirostris populations diverged into two groups after the construction of the Three Gorges Dam (Li, 2007). Moreover, natural linkage between the Huai River system and the Yangtze River system was also isolated after the construction of Sanhe sluice in 1953. How these changes in hydrology influence the genetic diversity and

| Animal culture and DNA extraction
Daphnia similoides sinensis was collected from water bodies located in the middle and lower reaches of the Yangtze River, belonging to water systems of the Yangtze River and Huai River (Table 1). In the laboratory, animals were identified (Benzie, 2005;Jiang & Du, 1979) and then monoclonally cultured in an intelligent light incuba-

| PCR amplification
Fourteen pairs of primers were used for the microsatellite markers ( Table 2). The COI gene was amplified with the LCO1490

| Electrophoresis, sequencing, and data analyses
The PCR amplification products of the COI gene were checked by gel electrophoresis and then purified by the AxyPrep DNA Gel and mismatch distribution were used to detect the evolutionary history of D. similoides sinensis populations (Fu, 1997;Tajima, 1989) using   were mainly composed of three haplotypes, which had significant differences from the other five populations.  (Table 4). The maximum genetic distance appeared between the HBDH and JSH populations,

| Genetic diversity and
whereas the minimum was between the WHNH and JSTH populations.
Seven D. similoides sinensis populations located in the middle and lower reaches of the Yangtze River were grouped into three clusters ( Figure 3). Among them, cluster 1 dominated in the WHNH population, CH population, and JSTH population, and cluster 3 dominated in the JSH population, which is distributed in the Yangtze River basin.
However, cluster 2 dominated in the HBSH population and HBDH population which distributed in the Huai River basin, as well as in the HDL population which located in the lower reaches of the Yangtze river.

| Genetic differentiation of seven D. similoides sinensis populations located in the middle and lower reaches of the Yangtze River
Based on the microsatellite markers, there was lower F st between the HBSH population and other six populations (Table 5), especially between the HBSH population and HBDH population (0.027). The

| Phylogeography of seven D. similoides sinensis populations located in the middle and lower reaches of the Yangtze River
Because of the lower sample size of the HDL population, phylo- Tajima's test (D = −2.087, p < 0.05) showed that the JSTH population deviated from neutral evolution, whereas the other six populations did not deviate from neutral evolution.

| Genetic diversity and genetic structure of D. similoides sinensis located in the middle and lower reaches of the Yangtze River
Haplotype diversity (Hd) and nucleotide diversity (π) are two important parameters to study population genetic diversity of organisms (Tajima, 1983;Weir, 1990). Higher Hd and lower π values in the natural population means that the organism could expand after a period of lower population size and enhance the retention of new mutations (Crandall, Sbrocco, Deboer, Barber, & Carpenter, 2011;Grant & Bowen, 1998). In this study, based on mitochondrial COI gene sequences, higher Hd and lower π values suggested that D. similoides sinensis located in the middle and lower reaches of the Yangtz River experienced a bottleneck in the process of population formation.
This phenomenon may be related to the rapid expansion of aquatic animal populations after the bottleneck effect and the quick accumulation of Hd, as well as periodic flooding events that occur in this region (Xu, Yu, & Ma, 2005). The Fu's Fs neutral test and Tajim's test suggested that the D. similoides sinensis JSTH population had experienced a bottleneck effect in the history. The JSTH population is located in Lake Taihu, which is part of the lower reaches of the Yangtze River. To improve water quality, two water transfer projects from the Yangtze River to Lake Taihu were conducted from 2002 to 2003 (Hu, Zhai, Zhu, & Han, 2008). However, the ecosystem in Lake Taihu became unstable after water transfers (Zhai, Hu, & Zhu, 2010).
These water transfer projects might have resulted in the JSTH bottleneck and affected the population structure of D. similoides sinensis in Lake Taihu.
In this study, seven D. similoides sinensis populations located in the middle and lower reaches of the Yangtze River were grouped into three clusters based on the microsatellite markers. According to the location of these populations, three clusters appeared to be related to geography, which cluster 2 was dominant in the Huai River basin whereas cluster 1 dominated along of the Yangtze River.
Moreover, the dendrogram (NJ/ME) based on the mitochondrial genes marker showed that six D. similoides sinensis populations were obviously clustered into two main clades, whereas there were three clades based on the microsatellite markers. One reason for the differing results between the mitochondria and nuclear data is significant differences in the evolutionary rate and level of polymorphism between different molecular markers (Bai & Zhang, 2014). The mitochondrial DNA have the characteristics of maternal inheritance (Avise et al., 1987), whereas the nuclear genes have higher mutation rates and are more appropriate for determining the genetic differences in organisms among different geographic populations (Al-Hamidhi et al., 2015;Selkoe & Toonen, 2006). The above results suggested that D. similoides sinensis located in the middle and lower reaches of the Yangtze River show significant genetic differentiation.

| Influence of geographic isolation on the phylogeography of D. similoides sinensis located in the middle and lower reaches of the Yangtze River
The influences of geographic isolation on the phylogeography of aquatic organisms have extensively been researched in the world. Machordom and Doadrio (2001) found that the geographic isola- Avise (1989) argued that the phylogeny of biogeographic patterns depends on the relationship between the phylogeny and geographical distribution of population. Population differentiation of organisms tends to be closely related to geographical distances, but recent population expansion and habitat fragmentation (Templeton, Routman, & Phillips, 1995) might affect the genealogy of species.

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

DATA ACCE SS I B I LIT Y
The sequencing data of Daphnia similoides sinensis in this study were deposited in DRYAD (https://doi.org/10.5061/dryad.66p5487).
F I G U R E 6 The observed pairwise difference (red line) and the expected mismatch distributions under the sudden expansion model (green line) based on the mitochondrial gene sequences of Daphnia similoides sinensis populations located in the middle and lower reaches of the Yangtze River