| Hypotrichous ciliates are the most specialized ciliated protozoa,with a wide distribution and rich species diversity,which is a popular group in taxonomic and biodiversity studies of ciliates.Due to the lag of effective barcode gene studies,the specific and intraspecies taxonomic definition of this taxa is still poorly understood.In this study,three species-complex,Euplotes vannus,Oxytricha granulifera,and Diophrys oligothrix,were used as experimental materials.Three nuclear gene fragments(SSU rRNA,ITS1-5.8S-ITS2,LSU rRNA)and mitochondrial genes(CO ?)were sequenced and analyzed,and morphometric analysis and morphological reobservation under the guidance of the reverse taxonomic thinking were performed with the purpose of exploring the taxonomic boundaries of species supporting the molecular clues.The results are as follows.1.Euplotes vannusThe results of five different geographical populations of E.vannus showed that there were significant genetic differences between Sanya population and other populations in the D1-D2 hypervariable region of LSU rRNA gene and CO ? gene sequence alignment.Especially for CO ? gene,the divergence of total gene substitution was as high as 18.7-21%,which was much higher than that of nuclear gene.There were differences in amino acids at multiple sites in amino acid sequence alignment,that was,non-synonymous substitutions of bases occurred in the corresponding codon at this site.At the same time,as for the molecular evolutionary tree constructed by LSU rRNA,CO? gene and amino acid,Sanya population was always an independent branch.The principal component analysis and pairwise test based on the data of 25 individuals and15 classical taxonomic traits in each population showed that the Sanya population was significantly different from other populations in three morphological characteristics:body width,oral area length,number of adoral zone of membranelles,and length of paroral membrane.Additionally,the morphological characteristics of Sanya population were observed again.Compared with other populations,the number of flower-annular extrusomes in dorsal cirrus was significantly increased in Sanya population.Therefore,both molecular and morphological implications suggested that the Sanya population represented differentiation at the subspecies level within E.vannus.2.Oxytricha granuliferaFive different geographical regions of O.granulifera research results showed that the population of O.atypica nov.spec.,which was isolated from the O.granulifera Saudi populations,in spite of the high similarity between the SSU rRNA genes in both groups.The additional fragmentation of anlage V during the ontogeny resulted in the formation of typical 7 posterior ventral cirri and stable differences between O.granulifera and other populations in CO ? gene.Therefore,it was established as a separate morphologic species.In the clustering relationship between the SSU rRNA and LSU rRNA phylogenetic trees,the Tibetan population also showed differences from other populations.The re-observation of the Tibetan population,whose gene sequences were submitted by the predecessors,showed that,compared with the type specimen population and the Austrian population,there were more dorsal bristles in each dorsal kinety.At the same time,the results of ANOSIM analysis showed that there were significant differences in body length and the number of left/right marginal criir.Consequently,the Tibetan population was tentatively named O.cf.granulifera.The population of Donghu in Wuhan was smaller than other populations in the proportion of the length of the oral area and the proportion of the body length of the oral area,and the number of the left and right marginal criir was smaller than other populations.The results of gene sequence analysis showed that there were genetic differences between Wuhan Donghu population and other populations in the molecular evolutionary tree constructed by nuclear gene,CO ? gene and amino acid.In consequence,the Donghu population of Wuhan was probably a subspecies within O.granulifera.There were a lot of nucleotide differences in the V4 region of SSU rRNA sequence between Wuhan Botanical Garden population and the type population,and the molecular evolutionary tree constructed by SSU rRNA sequence also indicated that there were genetic differences between this population and other populations.Therefore,the population of Wuhan Botanical Garden was probably a cryptic species.3.Diophrys oligothrixFour D.oligothrix populations from different geographic regions were not a single line in the molecular evolutionary tree constructed by three nuclear genes,CO ? gene and its amino acid sequence.At the same time,the Chinese fishing village population and other populations in the ITS2 region,D1-D2 region of LSU rRNA,CO ? gene sequence comparison,the nucleotide difference was obvious.There were significant differences in body length,the length of the oral area,and the number of dorsal bristles in the second row and the third row between the two populations.So the Chinese fishing village population was probably a subspecies within this species.In conclusion,we have carried out a species-level taxonomic studies on hypotrichous ciliates using the integrated multiple marker gene sequencing and morphological measurement methods for the first time.Our study showed that:(1)Genetic differentiation and even stable morphological differences were commonly existed among species under the traditional morphological classification.(2)The hypervariable region of nuclear genes(ITS2 region,D1-D2 region of LSU rRNA)and CO ? gene were effective in exploring the classification of the suspected species of hypotrichous ciliates.(3)Based on the clues generated by barcoding genes,the reverse taxonomy practices including morphological re-observation and morphometric analysis might find "new" morphological features distinguishing species-complex.Our study not only supplys a new patch of reliable barcoding sequences for the ciliates taxonomy but also provides case study for the species-level or below in ciliates. |
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