DNA-based Identification And Population Genetics Of Ramphogordius Sanguineus (Rathke,1799) (Nemertea: Heteronemertea)

1、Mapping the distribution of Ramphogordius sanguineus (Nemertea:Heteronemertea): a preliminary study based on DNA identificationThe heteronemertean Ramphogordius sanguineus is morphologically similar to several other species like Lineus ruber and Lineus viridis, and the identification of them is mostly based on coloration and the pattern of contraction when disturbed, which may not be always reliable. With each of these nominal species possessing numerous synonyms and having been, at some time, used as a name for the others, some records of these species are questionable and need to be clarified. In this study, we sequenced the barcoding region of COI gene for 129 specimens (identified as R. sanguineus according to external appearance) recently collected from 16 localities on coasts of Spain, China, Canada and Argentina. In conjunction with the sequences of 13 specimens from GenBank, a DNA based species identification is conducted by means of statistical parsimony and phylogenetic analyses. Our results show that:R. sanguineus is widely distributed along the Chinese coasts, and it is first recorded from waters of the northern Yellow Sea, the East China Sea and the South China Sea; the occurrence of R. sanguineus in Vancouver Island (Canada) is confirmed, which suggests that nemerteans reported as L. ruber or L. viridis from the Pacific Northwest may refer to this species; the specimen recorded as L. ruber from Chile should also belong to R. sanguineus; and R. sanguineus is also distributed on the Atlantic coast of South America (Argentina). In addition, present analyses reveal the occurrence of L. viridis in Qingdao, which is the first record of the species from Chinese waters.2、Genetic diversity of Ramphogordius sanguineus (Nemertea:Heteronemertea), an analysis based on the major non-coding region of mitochondrial DNA and nuclear rDNA ITSRamphogordius sanguineus, as is widely distributed in the world, is a fissiparous nemertean that is found beneath rocks and stones on sandy/muddy substrates as well as among mussels, oysters and other fouling organisms on hard substrates in the intertidal zone. Individuals can reproduce asexually from any part of the body and grow into adult. Previous studies on the species mostly focused on its taxonomy, physiology, regeneration and so on, few investigations were done for the population differention. In present study, we use the major non-coding region of mitochondrial DNA and internal transcribed spacers (ITS 1-5.8S-ITS2) to analyze the population genetic structure and historic dynamics of this species. Analysis on major non-coding region of mitochondrial DNA showed high haplotype diversity (Hd= 0.94±0.0096) and nucleotide diversity (π=0.044±0.022). All individuals from China, Canada and Spain were generated into two lineages (Lineage A and Lineage B), but neither lineage represented any population genetic structure. Individuals from the same region did not tend to cluster into a clade. When dividing all populations into three groups (China, Canada and Spain), no population genetic structure was detected (FCT=0.039,P>0.05) by AMOVA (analysis of molecular variance), which might be related to the uneven sampling and the small sample size of some populations. Besides, AMOVA analysis showed that the percentage contribution of within-population variation (55.53%) was greater than that of the inter-population variation (3.92%). Neutrality test Fu’s Fs showed that individuals from Lineage A experienced population expansion, while the individuals from Lineage B took the pattern of stable growth. It is likely that the two lineages evolved by allopatric differentiation, and the present overlapping distribution of the two lineages should be a result of the more recent expansion of lineage APresent analyses on ITS region revealed high intra-individual variation. We found that all copies of the 5.8 S rDNA within the same individual possessed three conservative three regions (M1, M2, and M3) and had the typical secondary structure and the G+C content for ITS was high (over 60%), which suggested that the sequenced ITS fragments were not pseudogenes. Consistent Bayesian phylogenetic tree could not be generated by ITS dataset, which may be because of the high intra-individual variation for this marker. Because asexual reproduction occurs frequently, gene conversion and unequal crossing over may be low in this species, which can thereby results in the occurrence of incomplete concerted evolution.