Taxonomy,Morphogenesis And Molecular Phylogeny Of Hypotrichous Ciliates

Ciliates are a large group of unicellular eukaryotes,which is the highest specialized group in Ciliophora.Ciliates are an important part of biological communities which playing an important role in the matter cycling and energy flowing of natural ecosystems.The subclass Hypotrichia locate at the highest stage of the differentiation of unicellular eukaryotes,being critical and special in phylogenetic researches,and are of great significance in the fields of cytology,genetics,eukaryote origin and evolution.Although thousands of ciliate species have been reported and described,there are still a mass of unknown,half-known and even blank populations that need to be identified and discovered.In this work,hypotrichous ciliates are collected from North China,Guizhou province and Iceland,and the morphology,morphogenesis and the molecular phylogeny was conducted by using morden methods.A total of 12 genera and 13 species of Hypotricha ciliates were isolated and identified.The main results as follows:1.MorphologyIt is found for the first time that Bakuella(Bakuella)marina(type species of Bakuellidae)has colorless cortical granules.The abnormal individuals of the indoor cultured population of Oxytricha granulifera granulifera were found and described.The morphological characteristics of four halophilic populations(B.(B.)marina,Cladotricha niesseniae,Cladotricha australis,Diophrys oligothrix)are described.Simultaneously,morphology of 8known species were also descripted and compared with other populations,such as Urostyla grandis,Pseudourostyla cristata,Paraurostyla weissei,Gonostomum strenuum,Rigidohymena candens,Gastrostyla steinii,Stylonychia bifaria,Uroleptoides magnigranulosus.The morphological characteristics of B.(B.)marina and the normal and abnormal forms of O.granulifera granulifera are as follows:(1)B.(B.)marina: Size in vivo 150–200 × 35–60 ?m.Cortical granules colorless.Three distinctly enlarged frontal cirri,more than one buccal cirrus,five to ten frontoterminal cirri,midventral complex comprised of midventral pairs and midventral rows,transverse cirri present,one left and one right marginal cirral row,caudal cirri absent.Collected from hypersaline water(suitable salinity approximately 50‰).(2)Normal form of O.granulifera granulifera: Body flexible and slightly contractile,elliptical in shape.Body size 90–130 × 30–45 ?m in vivo.The arrangement of 18frontal-ventral-transverse cirri in a stable 8:5:5 pattern.Undulating membranes in Oxytricha pattern.Constantly two globular to ellipsoid macronuclear nodules.Colorless cortical granules.Five rows of dorsal bristles arranged in typical Oxytricha pattern.(3)Abnormal form of O.granulifera granulifera: Size in vivo 115–140 × 20–25 ?m.Body slender with both ends rounded,slightly dorsoventrally flattened.One to four ellipsoidal macronuclear nodules.Cortical granules colorless and spherical.Buccal cavity short and narrow,undulating membranes absent.Three to four frontoventral,two to three postoral ventral cirri,the number and position of other cirri are stable.The mainly differences between abnormal and normal individuals are the size of the body,body shape,the length of adoral zone and the presence or absence of undulating membranes.Morphological characters of the other 3 halophilic species are as follows:(1)C.niesseniae: Body elliptical,flexible but not contractile.Size in vivo about 95 × 20?m.Adoral zone occupies about 50% of body length in stained specimens.Two macronuclear nodules.Cytoplasm colorless,usually containing food vacuoles,lipid droplets and minute crystals.Cortical granules lacking.Three slightly enlarged frontal cirri,two to four buccal cirri,three dorsal kineties arranged in Gonostomum-pattern,three caudal cirri,one each at the end of kineties 1–3.Highly halophilic,tolerance to the salinity of 60–230‰.(2)C.australis: Body fusiform,size 100–125 × 20–30 ?m in vivo.Adoral zone about30–40 ?m long.Fourteen to twenty-eight macronuclear nodules.Contractile vacuole usually locating near posterior end of adoral zone.Two buccal cirri,three frontoventral cirral rows.Highly halophilic,suitable salinity about 50‰.(3)D.oligothrix: Cell oval or elliptical,size 55–60 × 30–40 ?m in vivo.Adoral zone composed of 33 membranelles.With 14–15 cirri in the ventral side,2–3 marginal cirri.With4 dorsal kineties,3 caudal cirri.Halophilic,tolerance to the salinity of 50–150‰.Feeding on microalgae(diatoms).Usually crawl slowly on the sedment.2.Morphogenesis(1)The ontogenetic process of B.(B.)marina is revealed for the first time,and the main characteristics are as follows: 1)the oral primordium in both proter and opisthe develops de novo;2)frontal-ventral-transverse cirral anlagen are secondary mode.Anlage I contributes the left frontal cirrus,anlage II forms the middle frontal cirrus and the buccal cirri,anlage III produces the right frontal cirrus and the parabuccal cirri;3)anlage IV and on average next six anlagen form the midventral pairs,anlagen n-1 and on average previous six anlagen develop the midventral rows,anlage n forms the frontoterminal cirri.Usually,seven rightmost anlagen each develop one transverse cirrus,respectively;4)The formation of the marginal rows and dorsal kineties proceeds in the usual way,the macronuclear nodules fuse to a single mass during middle stage.(2)The cytogenesis of normal form of O.granulifera granulifera is very similar to that of the population discovered by Foissner and Adam in 1983.The main morphogenetic features are: 1)the oral primordium of the opisthe originates de novo and the parental adoral zone of membranelles remains unchanged;2)six streaks of frontal-ventral-transverse cirral anlagen are formed in both proter and opisthe;3)the marginal rows and the nuclear apparatus develop in the usual manner;4)dorsal kineties 1–3 develop within the parental kineties,dorsal kinety 4 originates from kinety 3 and kinety 5 originates dorsomarginally.(3)The morphogenetic process and conjugate reproduction of abnormal forms of O.granulifera granulifera were not observed.(4)The opisthe of C.niesseniae,the formation of the oral primordium begins with apokinetal proliferation of basal bodies posterior to the buccal vertex,the parental adoral zone of membranelles remains unchanged;the undulating membranes anlage is probably formed from dedifferentiation of the parental structure;the parental partial cirri might participate in the formation of frontoventral cirral anlagen.(5)The cytogenetic characteristics of C.australis are consistent with that of the genus Cladotricha,the parental adoral zone of membranelles remains unchanged;undulation membranes anlage of the proter is derived from the dedifferentiation of the old structure,undulation membranes anlage is formed on the right anterior side of the oral primordium in the opisthe,frontoventral cirral anlagen are formed in the secondary mode.Marginal cirri and dorsal kineties anlagen are formed by redifferentiation of the old structures.(6)During the middle and late morphologenetic stages of D.oligothrix,the macronuclear nodules fuse to a single mass;in the late stage,five anlagen developed into a typical cirri pattern of 3:2:2:3:1,and then evolved into the frontal-ventral-transverse cirri of the proter and opisthe,respectively;dorsal kineties anlagen being formed within each row,associated with three caudal cirrus at the right dorsal kinety rear end.3.Molecular phylogenyBased on the SSU rRNA gene sequence,the phylogenetic analysis of B.(B.)marina and O.granulifera granulifera were carried out,and their taxonomic status and phylogenetic relationship were discussed.(1)B.(B.)marina is the type species of the genus Bakuella and the family Bakuellidae.Since it was reported in 1976,there has been a lack of molecular data for a long time.This paper supplemented the 18 S rRNA gene sequence of this species.The phylogenetic tree based on 18 S rRNA shows that the family,genera and subgenera of Bakuella are all non-monophyletic,and this result was confirmed by AU test.(2)The normal and abnormal forms of O.granulifera granulifera are clustered into one branch on the phylogenetic tree,which supported that they were the same species.(3)The SSU rRNA gene of 11 species(B.(B.)marina,U.grandis,P.cristata,P.weissei,O.granulifera granulifera,C.niesseniae,R.candens,G.steinii,S.bifaria,U.magnigranulosus,D.oligothrix)were sequenced,which enriched the sequence information of ribosomal small subunit gene information of the groups involved.