Low Divergence of <i>Clonorchis sinensis</i> in China Based on Multilocus Analysis

<div><p><i>Clonorchis sinensis</i>, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of <i>C. sinensis</i> in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, <i>act</i>, <i>tub</i>, <i>ef-1a</i>, <i>cox1</i>, <i>cox3</i>, <i>nad4</i> and <i>nad5</i> [4.986 kb]) was employed to explore the intra-species genetic construction of <i>C. sinensis</i> in China. Two hundred and fifty-six <i>C. sinensis</i> isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in <i>cox1</i>, <i>cox3, nad4</i> and <i>nad5</i>, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of <i>C. sinensis</i> infection in mummified desiccated feces, these data point to an ancient origin of <i>C. sinensis</i> in China. In conclusion, we present a likely phylogenetic structure of the <i>C. sinensis</i> population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking <i>C. sinensis</i> infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of <i>C. sinensis</i>.</p></div

Divergence and differentiation among geographic distributions.

a<p>K_ST values are displayed above the diagonal and represent the weighted measure of the ratio of the average pair-wise differences within groups to the total average pair-wise differences.</p>b<p>S_nn values are displayed below the diagonal in bold and represent the proportion of nearest neighbors in sequence space that are found in the same group. Significance levels for K_ST and S_nn were assessed using permutation tests, with 1000 permutations:</p><p>ns = non-significant, ^*0.01**0.001***p<0.001.</p>c<p>Dxy values are displayed at the bottom of the diagonal and represent the minimum estimate of the number of nucleotide differences per site between groups.</p

Summarized archaeological evidence of <i>C. sinensis</i> eggs in desiccated fecal remains in mummies according to archived archaeology studies.

<p>Summarized archaeological evidence of <i>C. sinensis</i> eggs in desiccated fecal remains in mummies according to archived archaeology studies.</p

Phylogram constructed from UPGMA analysis of p-distances based on concatenated sequences of the ITS1 locus using global penal isolates (n = 288, <i>C. sinensis</i>: 271, <i>O. viverrini</i>: 7, <i>O. felineus</i>: 10).

<p>The fixed cluster I (red) is composed of 234 isolates from 17 provinces in China and 14 isolates from Korea and Japan. Cluster II (light blue) is composed of 16 isolates from Henan, Shannxi, Zhejiang, Guangxi, Anhui and Hubei province. Cluster III (light red) is composed of 6 isolates from Henan province and an ancient corpse from Hubei province in 176 BC. Seventeen <i>O. viverrini</i> (purple) and <i>O. felineus</i> (green) were used as outgroups. The percentage replicates are 1000 replications. The evolutionary distances were computed using the Maximum Composite Likelihood method.</p

Polymorphism summary and tests for neutral evolution in each locus.

<p>n.a: not applicable.</p

Divergence and differentiation among sub-populations.

a<p>K_ST values are displayed above the diagonal and represent the weighted measure of the ratio of the average pair-wise differences within groups to the total average pair-wise differences.</p>b<p>S_nn values are displayed below the diagonal in bold and represent the proportion of nearest neighbors in sequence space that are found in the same group. Significance levels for K_ST and S_nn were assessed using permutation tests, with 1000 permutations: ns = non-significant, ^*0.01**0.001***p<0.001.</p>c<p>Dxy values are displayed at the bottom of the diagonal and represent the minimum estimate of the number of nucleotide differences per site between groups.</p

Locations where wild cats, dogs and fishes were captured.

<p>Field sites are shown in relation to the ranges of the 17 proposed provinces (marked with red and purple) in mainland China. International borders and major rivers are also shown in the figure.</p

Divergence and differentiation among different hosts.

a<p>K_ST values are displayed above the diagonal and represent the weighted measure of the ratio of the average pair-wise differences within groups to the total average pair-wise differences.</p>b<p>S_nn values are displayed below the diagonal in bold and represent the proportion of nearest neighbors in sequence space that are found in the same group. Significance levels for K_ST and S_nn were assessed using permutation tests, with 1000 permutations: ns = non-significant, ^*0.01**0.001***p<0.001.</p>c<p>Dxy values are displayed at the bottom of the diagonal and represent the minimum estimate of the number of nucleotide differences per site between groups.</p

Neighbor-joining tree showing the phylogenetic relationships of the <i>C. sinensis</i> isolates included in this study (n = 256).

<p>All isolates were delineated into three major groups: the cluster A (n = 225) complex, cluster B (n = 19) and cluster C (n = 12). Geographically unique clusters were detected within the dendrogram tree: Henan (purple), Guangxi (green), Guangdong (blue) and Shandong (red). Isolates collected from fish and dogs are marked with symbols for fish and dog. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replications) are indicated. The evolutionary distances were computed using the Maximum Composite Likelihood method and are presented in units of the number of base substitutions per site.</p

Divergence and differentiation among water bodies.

a<p>K_ST values are displayed above the diagonal and represent the weighted measure of the ratio of the average pair-wise differences within groups to the total average pair-wise differences.</p>b<p>S_nn values are displayed below the diagonal in bold and represent the proportion of nearest neighbors in sequence space that are found in the same group. Significance levels for K_ST and S_nn were assessed using permutation tests, with 1000 permutations: ns = non-significant, ^*0.01**0.001***p<0.001.</p>c<p>Dxy values are displayed at the bottom of the diagonal and represent the minimum estimate of the number of nucleotide differences per site between groups.</p