Phylogenetic Analyses For The Mitochondrial Genomes And Transcriptomes Of Major Spider Mites

Tetranychidae belongs to Arthropoda,Chelicerata,Arachnida,Acari,Acariformes,Trombidiformes,Prostigmata,Eleutherengona,Raphignathae,Tetranychoidea,many of which are economic damaging mites.The potential diagnostic morphological characters of spider mites are limited and often exhibit great phenotypic flexibility between slides,which make it difficult to identify the spider mite species.These problems demonstrate the need for combining morphological and molecular approaches to identifying the spider mite species.However,the shortage and occasional unreliability of sequences in public databases restricts these sequences as molecular diagnostic tools.Worldwide Tetranychus urticae represents one of the most polyphagous arthropod herbivores.Some research about T.urticae has been also finished in our country,but T.urticae is not the only economic damaging mite.With seldom research,seldom useful DNA markers of the economic damaging mites in our country have been sequenced.Except the worldwide green morph of T.urticae,there is a red morph of T.urticae in our country.With the same morphological characters,they have partial hybrid sterility leading to identification controversy. Amphitetranychus viennensis was allocated in the genus Tetranychus before.It departed from Tetranychus and was allocated as a new genus Amphitetranychus.However,this new genus has seldom molecular basis leading to identification controversy.Some one still allocate it to Tetranychus.In view of the above problems,Long-PCR was performed to amplified the whole mitochondrial genomes of Tetranychus urticae red morph,T.urticae green morph,T. kanzawai,T.ludeni,T.malaysiensis,T.phaselus,T.pueraricola,T.truncatus and Amphitetranychus viennensis.Next generation sequencing was performed to get the sequences.After annotation,the substitution patterns,evolutionary divergence,evolutionary patterns,substitution saturation of the mitochondrial genomes were compared and the evolution relationships were established by using mitochondrial genome sequences. Besides,the phylogenetic relationship of Chelicerata mitochondrial genomes was established.The transcriptomes of T.kanzawai,T.ludeni,T.malaysiensis,T.phaselus,T. pueraricola,and T.truncatus were sequenced.After get the clean data,the transcriptomes were assembled.The homologous genes were pick to establish a phylogenetic tree. According to this tree,the dN/dS values of the homologous genes were calculated.The SSR of the transcriptomes were pick to establish a SSR database.The main conclusions are as follows:1)The mitochondrial genomes of spider mites are 13000 bp in length.Its relatively small size is primarily due to intensive mitochondrial genome and the significant size reduction of the A + T-rich region and tRNA.The gene order is the same in all mitochondrial genomes.However,this gene order is very different from that in other Acari and Cheliceratas.Twenty genes are encoded on the majority strand(7 PCGs,11 tRNA and2 rRNA),whereas the others are encoded on the minority strand(6 PCGs and 11 tRNA). The different spider mites have similar relative synonymous codon usage,codon usage pattern and base composition at each codon.The J-strands of the genus Tetranychus and Amphitetranychus mitochondrial DNAs exhibit adverse AT-skews and GC-skews than the J-strands of the genus Panonychus have positive GC-skews.2)Most tRNA genes in the spider mite mitochondrial genomes lose the D-arm and/or the T-arm.Except some tRNA genes,the secondary structures of the tRNA genes are conserved.All the 3 bp anticodon of the trnI genes in different spider mite mitochondrial genomes are flanked by 3 bp down-stream,which implies that this anticodon loop pattern is universal among trnI genes of the Tetranychidae mitochondria.3)Many tests of the homogeneity of substitution patterns between mitochondrial genome sequences reject the homogeneity hypothesis,which implies the existence of heterogeneity between mitochondrial genome sequences.The disparity index of heterogeneity are great between Panonychus and other spider mite genus,which probably caused by the adverse AT-skews and GC-skews.4)The diversity distance between the green and red morph of T.urticae is limited and smaller than that between spider mite species,which implies the green and red morph of T. urticae are the same species.The diversity distances between the genus Amphitetranychus and Tetranychus are larger than that among the genus Tetranychus,which supports the allocation of Amphitetranychus as a single genus.5)The transversion provides more mitochondrial DNA diversity than transition.The saturation of transversion and transition are rejected,which indicates the mitochondrial DNA sequences are pleasurable for phylogenetic analyses.The mitochondrial genome DNAs are used to establish the spider mite phylogenetic tree that reflect the spider mite phylogenetic relationships.6)The phylogenetic relationship of Chelicerata is established by the amino acid sequences of all the mitochondrial genes with the outgroup of Merostomata.The topologies of the ML and BI trees with high suppreting vallues are very similar.All the species of Chelicerata are allocated as a monophyletic group.Nearly all the orders are allocated as single monophyletic groups respectively,but Amblypygi and Uropygi are allocated together as a sister group.This probably implies that Amblypygi and Uropygi have closed relationship or the completed mitochondrial genome sequences of Amblypygi and Uropygi are limited to reconstruct their relationships.Pycnogonida is a class of Chelicerata,but Pycnogonida is not allocated as a separate group from Arachnida,which implies the ancestors of Pycnogonida and Arachnida separated very early.The spider mites are allocated together as a monophyletic group with a long branch,which indicates the mitochondrial DNA of the spider mites have a fast rate of evolution.7)The relationships of Chelicerata species are reconstructed by each PCGs' amino acid sequences,first plus second codon sequences and nucleotide sequences.In addition,the relationships of Chelicerata species are also reconstructed by the nucleotide sequences of rrnL and rrnS genes.However,the relationships of Chelicerata reconstructed by single gene fall to represent the real relationships of Chelicerata.A single gene probably does not have enough signals to reconstruct a relationship of high taxa.8)The transcriptomes of T.kanzawai,T.ludeni,T.malaysiensis,T.phaselus,T. pueraricola and T.truncatus have limited different in gene number.The phylogenetic tree reconstructed by the homologous genes are very similar to that reconstructed by the mitochondrial genomes in topologies.Most dN/dS values of homologous genes are less than 1,but some dN/dS values of T.kanzawai and T.pueraricola homologous genes are larger than 1.The EST-SSRs are also searched and the most common SSR types are A/T,AG/CT,AAC/GTT and ATC/ATG.