| Sipunculans,commonly called as sandworms,belong to the Sipuncula phylum,which is protostoma with true coelom.The phylum is rich in varieties with high conomic value.Hainan surrounded by the sea with many harbors,is famous for its abundant sipunculans resources,but there are few in-depth studies on the germplasm resources of sipunculans in Hainan being conducted at the moment.Siphonosoma australe is one of the common sipunculans with high economic benefits in Hainan,but the research on its coelomocytes and oocyte is also less.For this reason,we collected three common species(Sipunculus nudus;Phascolosoma esculenta;Siphonosoma austral)of sipunculans from Danzhou,Lingao,Puqian,Wenchang and Sanya in Hainan for the analysis of morphology and molecular.Moreover,the microstructure and ultrastructure of coelomocytes and oocyte of Siphonosoma austral were studied.The results as follows:(1)Two mitochondrial DNA markers(COI and CYTB)and one nuclear DNA marker(ITS)were used to construct the phylogenetic tree and haplotype network diagrams,which were significantly separated the three clades and each clade was corresponding to a single species of sipunculans.The result indicated that three molecular markers had good application to classify genus of sipunculans.(2)Discussion on taxonomic status of the Siphonosoma austral: in the morphological classification,Siphonosoma austral of Siphonosoma and Sipunculus nudus of Sipunculus belonged to the Sipunculidae.The Siphonosoma and the Sipunculus were sister genura in Sipunculidae.While in the phylogenetic tree,the Siphonosoma austral was closer to the Phascolosoma esculenta(belonging to the Phascolosomatidae)than to the Sipunculus nudus.It suggested that molecular classification and morphological classification were inconsistent,and molecular classification indicated taxonomic status of Siphonosoma austral might be an independent new family.(3)In the phylogenetic tree constructed by three DNA markers of Siphonosoma austral from Wenchang and Sanya,the molecular sequences of Siphonosoma austral could be divided into two clades: one included the sequences of Sanya partical samples(N1-N3,N5)and all samples from Wenchang,and the other included variant sequences from Sanya samples(N4,N6,N7),which indicated that there were cryptic species in Sanya.Moreover,the development of gell cell in this part of specific samples was earlier than that of other individuals,which probablely resulted from expansion of Xisha-island population in tropic sea area with the characteristic of early sexual maturity.(4)Population differentiation and affinity geography of The Sipunculus: from perspective of morphological analysis,four different geographical populations of Sipunculus nudus from Sanya,Wenchang,Puqian and Danzhou,presented the trend that the lengths of intestinal caecum increased from south population to north population,meanwhile the developed degree of Pulishi canal decreased.But the genetic structure of Sanya population is more complex.There were two groups in the COI phylogenetic tree,one group owning the varible intestinal caecum and Pulishi canal on different development-level,which was called the "Sipunculus nudus-like " group whose marker variation of genetic sequence was small.COI sequence of the other group was different from the sequences of "Sipunculus nudus-like " group.Morphological features of the other group owned developed Pulishi canal and no intestinal caecum,so it was identified as the Sipunculus robustus according to morphology.In the "Sipunculus nudus-like ",there was a small number of individuals with intestinal caecum degeneration in varying degrees(even without intestinal caecum),which were speculated to be hybrids from the "Sipunculus nudus-like " group and the group of Sipunculus robustus.Sanya is the birthplace of the agglomeration,differentiation,hybridization and diffusion of the Sipunculus in Hainan.(5)Population differentiation of Phascolosoma esculenta in Hainan: Compared with the two geographic populations of west Lingao and east Wenchang in Hainan,the appearance of large individuals of Phascolosoma esculenta have a high proportion in west population.but the differences in anatomical structure and molecular sequence of two geographic populations are the least among the populations of three species sipunculans,The result suggested that the population differentiation of Phascolosoma esculenta is small.(6)Observation of coelomocytes of the Siphonosoma austral: coelomocytes contained erythrocyte,osinophilic granulocyte,basophilic granulocyte,plate cell and urn cell complex.Most of the mature erythrocytes are concave-shaped.In the early stage of germ cell development,a large number of erythrocytes with concave-shaped were found in the coelom,suggesting that the germ cells transform from erythrocytes or that the erythrocytes were the origination of germ cells.The urn cell complex adheres to cells,and we have found that urn cell complex can phagocytosis small particles such as bacteria or cell debris in the body cavity.(7)The oogenesis of Siphonosoma austral could be divided into five stages: At the early growth stage,the oocytes less than 30μm were concave-shaped,and the surface of the oocytes was vesiculose,lumply and corrugated;At the late growth stage,the surface of30-40μm oocytes become smooth and unfolded,and the vitelline membrane appears with a thickness of 1μm;At the early mature stage,oocytes were 60-120μm in diameter,forming a spherical shape.Golgiosomes and yolks increased.The nucleus was enlarged.Some of the chromatin formed clumps of high electron density scattering in the nucleus;At the late mature stage,oocytes were 120μm in diameter,the membrane pore is exposed on the membrane surface.(8)The reproductive cycle of Siphonosoma austral in Hainan is from April to August,and the peak is in May and June.Oocytes begin to appear in early March,remain dense in the coelom from April to July,decrease in density in August and disappear in October. |
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