The Evolutionary Study On Monogeneans Using Mitochondrial Genomes

Monogeneans (Platyhelminthes, Monogenea) are important groups for classificatory and phylogenetic studies. To date, the evolutionary origin of monogenean is still controversial based on evidences from both morphological characters and molecular data. The mitochondrial (mt) genomes of metazoan have been widely used in evolutionary studies due to their small size, rapid evolutionary rate and maternal inheritance. In this paper, mt genomes of nine representative monogeneans were firstly determined and described, and comparative mt genomic analyses were performed. Phylogenetic analysis of monogeneans based on mt genomes data was performed, hoping to resolve the controversial issues in monogeneans’phylogeney. In view of phylogenetic results, the evolutionary patterns of mt genomes of monogeneans were firstly investigated. Followings are the main results:Firstly, the complete mitochondrial genomes of nine representative monogeneans were determined and annotated for the first time. Gene content, protein-coding genes (PCGs), tRNA and rRNA genes, gene intergenic and overlapped regions as well as gene arrangement were comparatively analyzed in all available monogenean mt genomes. Results demonstrated that most characteristics of monogenean mt genomes were similar, with a few of their variation. As for PCGs, cox1 was the most conserved gene and nad2 was the most variable gene, which provided useful information in choosing suitable molecular markers for future population systematic and phylogenetic studies. There were obvious biases in codon usage of PCGs, U and A were prefer in codons. The isotypes of trnI gene which existed in tandem repeats were firstly found in the mt genome of Polylabris halichoeres, which might be caused by slipped strand mispairing in replication. The anti-codon variation (TCG to ACG) of trnR gene was found in the mt genome of Benedenia hoshinai. Major non-coding regions (MNRs) had different characteristics among different subclasses of Monogenea. The size and repetitive frequency of repeat units was the main difference, which might be used to infer phylogenetic relationship of high taxonomic groups. Overlapped regions between nad4L and nad4 genes were widespread, but intergenic sequences (116 bp and 208 bp) between nad4L and nad4 genes were found in two species (B. hoshinai and B. seriolae) of Benedenia. Considering these two genes were overlapped in Neobenedenia melleni, such intergenic sequences between nad4L and nad4 genes should be considered as a sympatric character of the genus Benedenia. Gene rearrangement rates were different among each subclass in Monogenea, the rate in Oligonchoinea and Polystomatoinea was obviously higher than that in Polyonchoinea. Alignments of mitochondrial gene arrangement suggested phylogenetic information. The gene arrangements between Oligonchoinea and Polystomatoinea were basically consistent if gene rearrangement“hot spots”were out of consideration.Secondly, combining the newly determined monogenean mt genomes and previous published sequences, the phylogenetic analyses of monogeneans was performed. The phylogenetic position and origin of Monogenea was firstly investigated from mt genomic view utilizing three representative groups of monogenean. The results robustly favored the Trematoda and Cestoda were sister-group, but rejected the monophyly of monogenean. A sister group relationship for the Oligonchoinea+Polystomatoinea was robustly inferred, which shows a closer phylogenetic relationship with Trematoda+Cestoda, but the Polyonchoinea could not be inferred as a monophyletic group. Basing on the reconstructed phylogenetic relationship of parasitic flatworms and special lifecycle of Polystomatoinea, the evolutionary origin of parasitism in parasitic flatworms was inferred to be from ectoparasitism to endoparasitism, at the same time, it suggested that host-switching was from aquatic vertebrates to terrestrial vertebrates.Third, the evolutionary pattern of mt genomes in monogeneans was inferred using comparative genomics method based on the results of phylogenetic analyses. The results were listed as following: 1) The AT contents of mt genomes were differential among different subclasses, which was consistent with the result of phylogenetic analysis; the base skew was differential only in Gyrodactylidae of Polyonchoinea. 2) The same mutation degree occurred to the leucine zipper motif of nad4L proteins in mitochondrions of Cestoda and Monogenea, which was different only in Gyrodactylidae; the preferred start codon (GTG) was identical in Cestoda and Trematoda, but different from Monogenea (ATA), and the preferred stop codons were coincident in all parasitic flatworms; the total codons and codon usage were differential among different subclasses; the evolutionary rate of PCGs in Monogenea was consistent with that in other parasitic flatworms, which could be applied to molecular taxonomy, population genetics and phylogeny. 3) Lacking of arms in tRNA genes were differential among most of subclasses, but similar between Oligonchoinea and Polystomatoinea. 4) Characteristics of mt MNRs showed better concordance between Oligonchoinea and Trematoda+Cestoda, but differentiation in different groups of Polyonchoinea. 5) Polyonchoinea showed relatively consistent gene arrangement with Cestoda and Trematoda, but it took place great gene rearrangements comparing with Oligonchoinea+ Polystomatoinea.In conclusion, the evolutionary patterns of mt genomes showed large similarities between Oligonchoinea and Polystomatoinea, such as gene arrangements, bases content and total codons, which were also supported by the phylogenetic analyses. The evolutionary patterns of mt genomes had not occurred to obvious differentiation due to different parasitic behaviors between Oligonchoinea and Polystomatoinea. The evolutionary patterns of mt genomes for different groups in Polyonchoinea gave rise to differentiation to different extent, such as they had respective characteristics in Gyrodactylidae and Capsalidae (bases content, characteristics of MNRs and overlapped genes etc.), and the phylogenetic analyses supported that it was paraphyly. Based on the present evidence, the traditional concept of monogeneans was challenged, and the class Monogenea might not represent a natural taxonomic group. Oligonchoineans and polystomatoineans should be combined together and consider as a new phylogenetic lineage of parasitic flatworms, but the phylogenetic position of each group in Polyonchoinea need further verify.