Study On The Systematics Of Frontonia Ciliates(Ciliophora,Protozoa)

Frontonia,the largest genus of the Oligohymenophorea(Ciliophora,Alveolata)the subclass Peniculia,is found in various habitats,including freshwater,soil,and marine.The genus members exhibit similar general morphology and form a wellrecognized taxonomic group.As one of the most studied genera,there are still problems within the genus such as poorly defined basic morphological descriptions of some species,unclear interpretation of internal systematic relationships,and insufficient DNA barcoding data.In this study,we used multigene(SSU r DNA,ITS1-5.8S-ITS2,LSU r DNA,Cox I)sequencing,phylogenetic analyses,and various morphological observation techniques and adopted seven species of Frontonia(F.paramagna,F.fusca,F.leucas,F.atra,F.apoacuminata,F.depressa,F.depressa,and F.sp.)as materials to explore several systematics issues of the genus.The main results of the study are as follows.1.Complementary descriptions were given for 2 species that were previously poorly defined.For the first time,the number of peniculi 3 in the oral region of F.depressa was clarified,and the size and contraction time of the contractile vacuole were complemented to give a new definition of the species.A high variation in the number of contractile vacuoles within the F.paramagna population was found,and the contractile vacuole characteristics in the definition of the species were updated.2.For the first time,multigene data for F.depressa have been added,and based on this,a new branch(Clade IV)has been discovered that is located at the base of all Frontonia clade,showing that the complexity of relationships within the genus has been underestimated.Clade II is the core clade in which F.paramagna and the type species of the genus F.leucas are located.This clade includes two small clades and is in a stable position in any phylogenetic tree.Clade I of F.fusca is still clustered with the family Parameciidae in all phylogenetic trees except for Cox I,while it is clustered with clade II in the Cox I phylogenetic tree.AU test topology shows that the phylogenetic position of this clade is uncertain.The clade III of F.atra and F.apoacuminata is sister to the other Frontonia clades and shows rejection of all marker genes except for Cox I.Therefore,the status of clade III is relatively certain and always clusters with the family Stokesiidae.3.The morphology of nematodesmata representative of three phylogenetic clades was revealed,and the nematodesmata of species from different clades of the molecular phylogenetic tree were found to be different,indicating that nematodesmata may be a new morphological perspective to explain the status of genetic clades within the genus.The nematodesmata of F.paramagna,F.leucas,F.atra,F.apoacuminata,and F.depressa were initially investigated by using Flutax and transmission section observation.The nematodesmata of F.paramagna and F.leucas in core clade II were mainly distributed in the right side of the oral region at the vestibular kineties(N-vk1-3),the left side of the oral region including the peniculi 3(N-p3)and the posterior oral region at the postoral kineties(N-pk),and the postoral suture extending to the end of the body;the left and right sides of the oral area were connected in a "wrapped" state.The distribution of the nematodesmata of F.atra and F.apoacuminata in Clade III was the same as that of the above core clade,but they were not connected in a "wrapped" state and had almost no extension at the postoral suture.The N-vk of F.depressa in the outermost clade IV contains only two rows,and the left and right nematodesmata are not directly connected but through the deep fiber network,and there is almost no extension at the postoral suture.4.DNA barcoding was used to reveal the possible existence of cryptic species differentiation in two morphospecies(F.fusca and F.depressa).A comparison of genetic distances for multiple genes revealed that all genetic distances were much greater than the criteria for intraspecific genetic distances in ciliates.Genetic differences between the F.fusca Ningbo Chunxiao Lake population and the Guangzhou population provided by NCBI were much smaller than those between the Dongshatan population and the Guangzhou population,especially in ITS(0.5% vs 7.1?8.9%)and Cox I(2.5% vs 28.2?28.9%);the differences between the two populations of F.depressa were reflected in all barcodes involved,such as ITS1(23%),ITS2(19.4%),LSU-D2(10.4%)and Cox I(16.1%?17.2%).This study refined the taxonomic definitions of two species,added 81 phylogenetic and DNA barcoding marker genes,and for the first time analyzed the phylogenetic significance of nematodesmata in the context of comparative phylogenetic relationships.It provides new information for the ?-taxonomy of ciliates,DNA barcode database accumulation,and a new morphological perspective for the resolution of complex phylogenetic relationships.