Genetic diversity of Phascolosoma esculenta base on 

mitochondrial cytb gene and d-loop region 

in the southeast coast of China

Abstract: Phascolosoma esculenta belongs to Sipunculoidea (order), Phascolosomalifo (class), Phascolosomatidae (family), Phascolosoma (genus). It is an endemic species of Sipunculoidea in China, which distributes in coastal tidal flats of the southeastern provinces. As one of the marine treasures, Phascolosoma esculenta is rich in protein and has a variety of unsaturated fatty acids and other nutrients. People call it "animal ginseng" and it is a characteristic economic aquatic creature of local residents. In recent years, the development and construction process of the southeast coastal areas of China has been accelerating, and environmental pollution, tidal flat reclamation, and overload harvesting have resulted in the sharp decline of germplasm resources of wild populations of P. esculenta in China, while the market price has been rising year by year. Therefore, the related resource protection, seedling breeding and enhancement has drawn enormous attention to the industry. However, there are few studies on the population genetics of this species. So it is necessary to study the genetic diversity of wild population of Phascolosoma esculenta for the rational development and sustainable utilization.

In this study, the cytb and dloop gene sequences of mitochondrial DNA were used as molecular markers to analyze the genetic diversity and genetic structure of a total of 150 samples from five natural populations of P. esculenta in Zhanjiang City, Guangdong Province (ZJ), Yangjiang City, Guangdong Province (YJ), Qinzhou City, Guangxi Province (QZ), Ningde City, Fujian Province (ND), and Wenzhou City, Zhejiang Province (WZ), which can provide corresponding basic information for artificial breeding and nursery of this endemic Phascolosoma species in China, and provide reference data for subsequent studies on the conservation of germplasm resources and sustainable exploitation of P. esculenta.

DNA was extracted from the body wall muscle of P. esculenta using a genome extraction test kit, primers were designed based on its published mitochondrial genes and synthesized by a thirdparty company. The mitochondrial cytb and dloop amplification fragments of P. esculenta through electrophoresis tests were selected for purification and sequencing, using a PCR amplification kit for PCR amplification of DNA samples. Sequencing analysis of 150 samples revealed that the cytb and dloop sequences showed small differences in the content of the four bases and all exhibited significantly higher A+T content than C+G content, with significant nucleobases bias, consistent with the published mitochondrial nucleobases characteristics of P. esculenta. In the five geographical groups, 118 and 158 polymorphic loci were detected for cytb and dloop sequences, respectively, defining 62 cytb haplotype types and 79 dloop haplotype types. Haplotypes of both genetic sequences were randomly dispersed among different populations, with some haplotypes common to multiple populations and some haplotypes specific to individual populations. Such a haplotype distribution could be biologically explained as the result of a combination of a recent exogenous genetic invasion event and subsequent genetic divergence in a geographic population. Analysis of genetic diversity parameters based on both cytb and dloop sequences showed high genetic diversity (cytb: 0.871 3-0.963 2; dloop: 0.942 50.981 6) and low level of nucleotide diversity (cytb: 0.032 4870.040 975; dloop: 0.033 247 0.046 151), with a high overall level of genetic diversity. Combined with the results of Tajima’s D statistic and Fu’ Fs neutrality tests, results of this analysis significantly deviated from the neutrality, through which we could infer that the P. esculenta population has recently experienced a rapid population expansion event, it was derived from a small breeding population to a large population in a relatively short period. The topology of the haplotype neighborjoining trees of cytb and dloop sequences of different populations of P. esculenta was simple and did not show a clear geographical genealogical structure; while the analysis of molecular variance (AMOVA) results showed that the intrapopulation genetic variation (cytb: 98.56%; dloop: 98.61%) was significantly higher than that between populations (cytb: 1.44%; dloop: 1.39%), indicating that the genetic variation of P. esculenta among different geographical groups was not obvious and there was no geographical segregation between different geographical groups. The consistency of the two results illustrated the absence of significant genetic differentiation and low genetic differences among the five geographic groups of P. esculenta.

In summary:1) Results of the analysis of two different sequences of the five geographic populations of P. esculenta in Zhanjiang City, Guangdong Province (ZJ), Yangjiang City, Guangdong Province (YJ), Qinzhou City, Guangxi Province (QZ), Ningde City, Fujian Province (ND), and Wenzhou City, Zhejiang Province (WZ), showed high levels of genetic diversity and low levels of nucleotide diversity, with a high level of overall genetic diversity. This feature was also reflected in other researchers’ studies on the genetic diversity of P. esculenta from other geographical populations along the southeast coast of China based on mitochondrial CO I and 16S rRNA gene sequences. Because the accumulation of gene diversity can be improved in a short time, but the accumulation of nucleotide variation takes a longer time. 2) Combined with the Tajima’s D statistic and Fu’s Fs neutrality tests, we can suggest that the P. esculenta population has recently undergone a rapid population expansion event; the genetic variation is not significant among different geographic groups, indicating that the five geographic groups of P. esculenta do not have significant genetic differentiation, and the genetic differences are small. The possible causes of this phenomenon are: ① P. esculenta lives in the intertidal zone along the coast with few geographical barriers. In its early life history, trochophore larvae and pelagosphera larvae disperse along the distance with the movement of sea water, resulting in gene exchange between different geographical populations. ② Due to the increasing maturity of artificial breeding technology and the expansion of breeding scale, many farmers purchase seedlings from various places and put them into local farms. However, the phenomenon of breeding population escape caused by the drainage of the farms often occurs, which makes the gene exchange between the geographical populations of P. esculenta frequent; ③ Because of the similar habitat conditions in the intertidal zone of the southeast coastal areas of China, the genetic variation direction of different geographical populations is indirectly affected. In addition, by combining the results of two algorithms of genetic distance, the genetic distance between ND (Ningde, Fujian) population and other populations is the closest, which may be related to the genetic confusion caused by the escape of artificial seedlings from local farms. The sampling point NiuMu Island located in the gulf is one of P. esculenta breeding bases in Changchun Village, Xiapu County of Mindong region. While the larvae of this species can not satisfy the requirement of aquaculture farmers, they will purchase a large number of larvae for breeding from Guangxi, Zhejiang provinces and other places. Due to the influence of tide, there must be escape in larvae of artificial breeding. This results in severe genetic confusion among local wild geographic populations.