Genetic Diversity of Daphnia pulex in the Middle and Lower Reaches of the Yangtze River

Increased human activities and environmental changes may lead to genetic diversity variations of Cladocerans in water. Daphnia pulex are distributed throughout the world and often regarded as a model organism. The 16S rDNA, cytochrome c oxidase subunit I (COI), and 18S genes were used as molecular marks. The genetic diversity and phylogeny of D. pulex obtained from 10 water bodies in the middle and lower reaches of the Yangtze River were studied. For 16S rDNA, COI gene, and 18S gene, the A+T content (65.4%, 58.4%, and 54.6%) was significantly higher than the G+C content (34.6%, 41.6% and 45.4%). This result was consistent with higher A and T contents among invertebrates. Based on the genetic distances of 16S rDNA and COI genes, the genetic differences of D. pulex from 10 water bodies located in the middle and lower reaches of the Yangtze River in China was minimal (0%–0.8% for 16S rDNA and 0%–1.5% for COI gene). However, D. pulex evolved into two branches in the phylogenetic trees, which coincided with its geographical distribution. Compared with D. pulex from other countries, the average genetic distance of D. pulex obtained from 10 water bodies in the middle and lower reaches of the Yangtze River reached 9.1%–10.5%, thereby indicating that D. pulex may have evolved into different subspecies.


Introduction
Cladocerans are important components of the food chain in aquatic ecosystems [1]. First, these organisms can feed on algae and improve water quality [2][3][4]. Second, cladocerans are predated by fish as food. Cladocerans undergo parthenogenesis in suitable environments and form large populations. However, sexual reproduction of Cladocerans occurs under bad conditions and fertilized eggs are produced. Daphnia pulex is a cosmopolitan species that is widely distributed in inland fresh waters, particularly in eutrophic waters [5][6]. To date, frequent human activities have led to environmental differences among lakes, such as variations in nitrogen and phosphorus concentrations in the sediment in the middle and lower reaches of the Yangtze River [7]. Eutrophication and the structure of the fish population may also have affected the population dynamics of D. pulex in these lakes [8][9][10].
Multiple methods are available for species identification and phylogeny reconstruction of crustaceans [11][12][13][14][15]. The 16S rDNA and the cytochrome c oxidase subunit I (COI) and 18S genes are more popular among these methods [15][16][17][18][19][20][21]. The classification of 16S rRNA and COI gene sequences were more convincing in Daphnia. The mitochondrial divergences of different Daphnia species are below 5% between North and South America [22][23][24] and between North America and Europe [25]. John et al. (2011) reported the gene sequences of D. pulex [26]. A few functional genes of crustaceans were widely studied [16,20,[26][27][28]. Benzie (2005) described the D. pulex complex, including D. pulex, D. pulicaria, and D. middendorffiana [6]. The different D. pulex complexes are distributed worldwide, and the species was studied as a model by many investigators [28][29][30][31][32]. Ceresa et al. (2012) investigated the intercontinental phylogeography of the D. pulex complex by analyzing the mitochondrial NADA dehydrogenase subunit 5 and the COI gene [29]. Some works in the literature showed that the genetic distance ranged from 5% to 14% for D. pulex complex [16,29,33]. Although the molecular phylogeny of D. pulex was extensively reported, the genetic differences of the D. pulex from China and comparison of species in China and those in other countries have not been reported.
In this study, the genetic difference among the D. pulex from 10 water bodies located in the middle and lower reaches of the Yangtze River and the genetic difference of the D. pulex between China and other countries were analyzed by amplifying and sequencing the 16S rDNA, as well as the COI and 18S genes. Our results could become an important evidence for the global phyletic evolution of D. pulex.

Sampling, identification and culturing
Field collection of Daphnia was carried out after obtaining permission from the Ministry of Environment, and the field studies did not include endangered or protected species.
The fertilized eggs of D. pulex were collected from the sediment of 10 water bodies located in the middle and lower reaches of the Yangtze River with a modified Peterson grab ( Table 1). The eggs were hatched in an intelligent lighting incubator (Ningbo Saifu, China) at 25°C. D. pulex was identified morphologically (Fig 1) under the microscope (Olympus, Japan) according to the methods of Jiang and Du [5] and Benzie [6]. For each water body, four individuals of D. pulex were selected by hatching different fertilized eggs. Monoclonal organisms were cultured in an intelligent light incubator (Ningbo Saifu, China) with 12 h light:12 h dark illumination at
To validate the predicted sequences of chemosensory genes, the PCR (Eppendorff, Germany) products obtained from genomic DNA of adult D. pulex were purified using the Axy-Prep TM PCR Cleanup Kit (Axygen) and sub-cloned into a T/A plasmid using the pEASY-T3 cloning vector system (TransGen, China) following the manufacturer's instructions. The plasmid DNA was used to transform to Trans1-T1 competent cells. The positive clones were checked by PCR and sequenced by GenScript (Nanjing, China).

Analytical procedure
According to the peak in SeqMan, the bidirectional sequencing of the nucleotide sequence was proofread by DNAStar to remove unreliable bases. The percentage of the detected sequence differences was obtained.
For each water body in the middle and lower reaches of Yangtze River, the sequences of the four D. pulex individuals were obtained. Unreliable bases were removed by SeqMan (DNAStar). A total of 517-539 valid bases for 16S rDNA, 522-527 valid bases for the COI gene, and 2335-2344 valid bases for the 18S genes were detected (Table 1). Other sequences that were used for analysis were downloaded from GenBank (Tables 2-4). In this study, the standard of the selected sequences was the similarity of the homologous sequence (over 80%) compared with the sequences from Genbank.
Multiple sequence alignment was performed with CLUSTALX (ref. ). DNAspV5 (ref.) was used to analyze the variation of sites among the sequences. The conversion/transversion and the genetic distance of interspecies were calculated with MEGA 6.0 (ref.). The genetic distances among sequences were calculated by the Kimura two-parameter model with 1,000 bootstraps. The maximum likelihood (ML) analysis, which used the GTR+G+I evolutionary model indicated by Modeltest version 3.7, was performed with MEGA 6.0 (ref.) and bootstrap resampled 1,000 times. In addition, we constructed phylogenetic trees via Bayesian inference in MrBayes 3.1.2 (ref.). This program was run for 10,000,000 generations, and sampling from the chain was performed every 10,000 generations. Initially, 25% of the trees were discarded as burn-in, and the 50% majority rule consensus tree was constructed from the remaining Bayesian trees after the posterior probability values for each node were calculated. To better reveal the genetic difference of D. pulex, the suitable outgroups were employed to construct phylogenetic trees. For 16S For 18S gene, D. magna (AM490278) and C. dubia (AF144208) were used as outgroups. Analysis of molecular variance (AMOVA) test was conducted by using Arlequin 3.5 [36].

Results
Genetic diversity of D. pulex from the middle and lower reaches of Yangtze River based on 16S rDNA The alignment of the 37 16S rDNA sequences identified 403 conserved sites, including 334 invariable sites, 69 variable sites, 9 single sites, and 60 parsimony-informative sites. Among the 16S rDNA sequences of the D. pulex from 10 water bodies located in the middle and lower reaches of Yangtze River, the average A, T/U, C, and G content was 32.6%, 32.8%, 13.6%, and 21.0%, respectively. The A+T content (65.4%) was significantly higher than the G+C content (34.6%). The overall transition/transversion ratio was 1.09. The genetic distances between sequences were calculated by the Kimura 2-parameter distance (0%-9.8%) and maximum likelihood estimate (0%-11.5%). The phylogenetic trees produced highly congruent tree topologies (Fig 2). The main divergences in the ML tree were in accordance with Note: D. pulex (GenBank accession: KJ775027) was obtained from Zhejiang province in China. those of the MrBayes and NJ trees (Fig 2). In the phylogenetic trees, the branches represented 99% support for the presumed biological species of D. pulex. D. pulex from 10 water bodies located in the middle and lower of the Yangtze River evolved into two branches in the NJ tree. One branch included D. pulex from Lake Donghu in Hubei Province, as well as Lake Shengjin, Lake Nanyi, Lake Chaohu, and Guohe River in Anhui Province. The other branch included D. pulex from a pond in Shanghai City, Lake Taihu, and Lake Hongze in Jiangsu Province, as well as Lake Qianlong, a pond, Lake Chaohu, and Lake Nanyi in Anhui Province. D. pulex from Lake Chaohu and Lake Nanyi was present in both branches, which coincided with its geographical locations. In addition, the D. pulex from 10 water bodies located in the middle and lower reaches of the Yangtze River and the D. pulex from abroad were clustered in two distant branches (Fig 2).
Genetic diversity of D. pulex from the middle and lower reaches of Yangtze River based on the COI gene The alignment of 26 COI sequences identified 487 conserved sites, including 433 invariable sites, 54 variable sites, 9 single sites, and 45 parsimony-informative sites. Among the COI sequences of the D. pulex from 10 water bodies located in the middle and lower reaches of Yangtze River, the average A, T/U, C, and G content was 23.5%, 34.9%, 20.1%, and 21.5%, respectively. The A+T content (58.4%) was significantly higher than the G+C content (41.6%). The overall transition/transversion ratio was eight. The genetic distances between sequences were calculated by the Kimura two-parameter distance (0%-11.3%) and maximum likelihood estimate (0%-11.4%). The main divergence in the ML tree was in accordance with that of the MrBayes tree and NJ tree (Fig 3). In the phylogenetic trees, the branches represented 100% support for the presumed biological species of D. pulex. The D. pulex from 10 water bodies located in the middle and lower reaches of Yangtze River diverged into two branches in the phylogenetic trees, which was consistent with the results of 16S rDNA sequence analysis. In addition, the D. pulex (GU595190) from Japan and the D. pulex from 10 water bodies located in the middle and lower reaches of Yangtze River were evidently different, with an average genetic distance of 10.5%.
Genetic diversity of D. pulex from the middle and lower reaches of Yangtze River based on the 18S gene The alignment results of 36 18S gene sequences identified 1963 conserved sites, including 1932 invariable sites, 31 variable sites, 20 single sites, and 11 parsimony-informative sites. Among the sequences of the 18S gene for the D. pulex from 10 water bodies located in the middle and lower reaches of Yangtze River, the average A, T/U, C, and G contents were 20.5%, 24.9%, 24.3%, and 30.3%, respectively. The A+T content (54.6%) was significantly higher than the G+C content (45.4%). The overall transition/transversion ratio was 2.5. The genetic distances between sequences were calculated by the Kimura two-parameter distance (0%-2.0%) and the maximum likelihood (0%-1.3%). The main divergence in the ML tree was in accordance with that of the MrBayes and NJ trees. The phylogenetic trees produced highly congruent tree topologies (Fig 4). In the phylogenetic trees, the branches represented 99% support for a presumed biological species of D. pulex. The D. pulex from Lake Chaohu in Anhui province (CH3), Lake Hongze in Jiangsu province, Hangzhou City in Zhejiang province (KJ775027), and Canada (AF014011) belonged to the same branch, whereas smaller differences were observed with the other D. pulex individuals. The average genetic distance was 0.45%-0.64%.  Tests on the genetic difference of D. pulex from the middle and lower reaches of Yangtze River Based on the sequences of 16S rDNA, COI gene, and 18S gene, the genetic differences of D. pulex were analyzed within lakes and between lakes in the middle and lower reaches of Yangtze River. Mann-Whitney Test showed that the genetic differences of the D. pulex between within-lakes and between-lakes were significant (COI gene: Z = -3.172, P = 0.002; 16S rDNA: Z = -3.096, P = 0.002; 18S gene: Z = -3.378, P = 0.001). Two-Sample Kolmogorov-Smirnov test showed the significant differences in both within-lakes and between-lakes (COI gene: Z = 1.789, P = 0.003; 16S rDNA: Z = 2.012, P = 0.001; 18S gene: Z = 2.012, P = 0.001). The box diagram of D. pulex genetic diversity also demonstrated significant differences between within-lakes and betweenlakes based on the sequences of 16S rDNA, COI gene, and 18S gene (Fig 5), which indicate that the genetic structure of D. pulex exhibiting differentiation among lakes. Within lakes, the genetic difference of D. pulex from Lake Chaohu (CH) was bigger than that of other lakes based on the sequences of COI gene and 18S gene, and the genetic difference of D. pulex from Lake Nanyi (XC) was bigger than that of other lakes based on the sequences of COI gene and 16S rDNA (Fig 5).

Analysis of molecular variance
Based on the sequences of 16S rDNA, COI gene and 18S gene, the Molecular Variance of D. pulex were analyzed to calculate the population genetics for each site in the middle and lower reaches of Yangtze River. The result suggested that the pairwise differences were greater among populations than within populations based on the sequences of 16S rDNA, COI gene and 18S gene ( Table 5). The AMOVA test showed that there were significant differences between two groups (middle reach and lower reach) based on 16S rDNA and COI gene (Table 6).

Discussion
For 16S rDNA and COI gene sequences of D. pulex from 10 water bodies located in the middle and lower reaches of Yangtze river, the A+T content (65.4% and 58.4%, respectively) was significantly higher than the G+C content (34.6% and 41.6%, respectively). Those results were consistent with the higher A and T contents among invertebrates [37][38]. Moreover, the overall transition/transversion bias of D. pulex based on COI gene (8) was obviously higher than those based on the 16S rDNA (1.09) and 18S gene (2.5).
Based on the genetic variation of the 16S rDNA and COI genes, the D. pulex from 10 water bodies located in the middle and lower reaches of the Yangtze River evolved into two branches, as shown in the phylogenetic trees. One branch included the D. pulex from Lake Donghu in Hubei Province, as well as Lake Shengjin, Lake Nanyi, Lake Chaohu, and Guohe River in Anhui Province. The other branch included D. pulex from a pond in Shanghai City, Lake Taihu and Lake Hongze in Jiangsu Province, as well as Lake Qianlong, a pond, Lake Chaohu, and Lake Nanyi in Anhui Province. The D. pulex from Lake Chaohu and Lake Nanyi in Anhui province were present in both branches, which coincided with its geographical distribution in the middle and lower reaches of the Yangtze River. Based on the sequences of 16S rDNA, COI gene and 18S gene of D. pulex, the AMOVA test also showed that there all were greater genetic differences among lakes than within lakes in the middle and lower reaches of the Yangtze River. And significant genetic differences between two groups (middle reach and lower reach) were showed based on 16S rDNA and COI gene of D. pulex. Then the genetic distances of D. pulex from 10 water bodies located in the middle and lower reaches of Yangtze River showed minimal divergence based on 16S rDNA (0%-1.0%), COI gene (0%-1.7%), and 18S gene (0%-0.9%), and all those differences were within the scope(<5%) of species [16,33,39]. These findings implied that the D. pulex from the lakes located in the middle and lower reaches of Yangtze River region should belong to the same species. In addition to further geographical distance, other environmental conditions, such as different climate, altitude, and fishery in the middle and lower reaches of the Yangtze River, may be important factors to the evolution of D. pulex.
Compared with the D. pulex from Canada (AF117817, GQ343275) [40,41] and Russia (JN874605, JN874606, and JN874607) [42], the genetic distances of D. pulex from 10 water bodies located in the middle and lower reaches of the Yangtze River reached 9.1%-9.6% based on 16S rDNA sequence. The genetic differences was obviously beyond the scope of a species (<5%) [16,33,39], and it indicated the presence of subspecies. Long-term geographic isolation may be the main reason for the evolution of the D. pulex in China and other countries. In addition, the average genetic distance between the D. pulex in Japan (GU595190) and the D. pulex in China reached 10.5% based on the COI gene sequence. The genetic distance was in the scope of the Daphnia complex (5%-14%). Thus, compared with the D. pulex (GU595190) in Japan, the D. pulex from China should belong to different subspecies or the D. pulex complexes [6,16,33,43]. On the other hand, the genetic distance of the D. pulex (GU595192) in Japan and in China was below 5%. We speculated that the D. pulex had same ancestor and evolved to different directions by natural selection in Japan and China. Although the average genetic distances of the D. pulex from Canada (AF014011) and from China were small (0.45%-0.64%) based on the 18S gene sequence, the difference was evident. In general, the evolutionary divergences of the D. pulex among different lakes located in the middle and lower reaches of the Yangtze River were minimal. However, the evolutionary divergence was relatively high compared with that of other countries. The global molecular phylogeny of D. pulex needs to be further studied and discussed. The intercontinental phylogeny of the D. pulex complex is extremely complicated. Based on the sequences of the mitochondrial dehydrogenase NADH 5 subunit and COI genes of 398 D. pulex individuals from five continents, Crease et al. (2012) concluded that 11 lineages of the D. pulex complex can be observed worldwide [29]. By studying the D. pulex complex from 12 Bolivian high-altitude lakes, the D. pulicaria group in North America was found to originate in South America, whereas these South American water fleas originated through reciprocal hybridization between different sexually reproducing parental lineages [44]. In the present study, based on the 16S rDNA sequence, the average genetic distances of the D. pulex from China and the D. parvula and the D. obtusa from Canada were 7.3% and 8.2%, respectively. Their differences belong to the scope of the D. pulex complex (5%-14%) [16,[29][30][31][32][33]45]. Benzie (2005) hypothesized that the main factor that led to the formation of species complexes between the D. pulex, D. pulicaria, and D. middendorffiana was their long-term coexistence in the same habitat, which resulted in the occurrence of interspecies complexes [6,16]. In the middle and lower reaches of the Yangtze River in China, the coexistence of D. pulex, D. galeata, and D. similoides was common in some lakes (e.g. Lake Donghu, Lake Taihu, and Lake Chaohu) [8][9][10]. Thus, the existence of species complexes among Daphnia species in these Chinese lakes was possible, and further investigation is needed.