| Objective:Some Gamasina species that parasitize the body surfaces of small mammals can directly or indirectly transmit a variety of zoonotic diseases.The scarcity of sequence data has greatly hampered the phylogenetic study of Gamasina species,resulting in the current taxonomic status of Gamasina’s different orders remaining controversial.In order to enrich the mitochondrial genome database of Gamasina,the mitochondrial genomes of three gamasid mites（Parasitus fimetorum,Eulaelaps huzhuensis,and Eulaelaps silvestris）in two families and two genera of Apodemus chevrieri ectoparasites were determined and analyzed for the first time in this study and combined with information from mitochondrial genomic data for sequenced species of Gamasina,to explore the evolutionary relationships among different taxonomic orders of Gamasina.Method:In this study,we used the PCR amplification method and high-throughput sequencing technology to sequence the three gamasid mites’mitochondrial genomes.Geneious Prime 2021.1.1 and Mito Z 2.3 was used to assemble and splice the sequenced data,and the genes were found and annotated based on NCBI,MITOS,t RNAscan-SE,and ARWEN.The base composition,protein-coding genes,relative synonymous codon usage,base substitution saturation,evolutionary rate,and gene rearrangements were systematically analyzed using various bioinformatics software.Meanwhile,maximum likelihood trees and Bayesian trees were constructed based on two datasets（PCGs+r RNAs and PCGs）to explore the phylogenetic relationships of Gamasina,respectively.Result:（1）The mitochondrial genomes of the three gamasid mites showed typical loop-closed double-stranded DNA of 14,619 bp（P.fimetorum）,14,872 bp（E.huzhuensis）,and 14,882 bp（E.silvestris）lengths,all encoding 37 genes,all with a clear AT preference in base composition.Codons ending in A/U are mostly preference codons,while codons ending in G/C have weaker preferences.（2）P.fimetorum has 1control region.Between adjacent genes were 12 gene intergenic regions and 9 gene overlap regions.The J-strand encodes 22 genes,and the N-strand encodes 15 genes.There are 11 protein-coding genes using ATN for the initiation codon,the remaining two protein-coding genes using GTG as the initiation codon,and four protein-coding genes ending with base T.A total of 23 mismatches occurred in 22 t RNA genes,and all t RNA genes except trn S1could fold into a typical cloverleaf structure.The most frequently used codon was UUA（Leu）.（3）E.huzhuensis has 2 control region.Between adjacent genes were 10 gene intergenic regions and 12 gene overlap regions.The J-strand encodes 23 genes and the N-strand encodes 14 genes.There were 12protein-coding genes using ATN for the initiation codon,only atp8 used GTG as the initiation codon,and three protein-coding genes ended with base T.A total of 30mismatches occurred in 22 t RNA genes,and only three t RNA genes（trn S1,trn S2,and trn D）could not form a cloverleaf-type structure.The most frequently used codon was UUA（Leu）.（4）E.silvestris has 2 control region.Between adjacent genes were 10gene intergenic regions and 12 gene overlap regions.Twenty-three genes were encoded by the J-strand and 14 genes were encoded by the N-strand.13protein-coding genes have ATN as the initiation codon and two protein-coding genes ending with base T.A total of 37 mismatches occur in 22 t RNA genes,and 20 t RNA genes have a typical cloverleaf structure,with only trn S1and trn S2missing the D arm.The most frequently used codon is UUA（Leu）.（5）Three gamasid mites protein-coding genes with the fastest and slowest evolutionary rates were the atp6gene and the cox1 gene,respectively.Eight protein-coding genes experienced negative selection.Codon analysis reveals natural selection pressure as a major factor in the codon preference of three gamasid mites.（6）Only species of the families Parasitidae and Diplogyniidae in Gamasina had no rearrangements in their mitochondrial genomes,while the remaining 9 families and 20 species had various degrees of rearrangements.Except for two species of the genus Eulaelaps in the family Haemogamasidae with the same type of rearrangement,the rearrangement patterns of the remaining 18 species in 8 families were different.The same derived gene clusters are shared among some species of the same family or genus.The rearrangements of Gamasina species occurred mostly near t RNA genes and control regions.Rearrangements in species of the family Varroidae was associated with t RNA genes only,while rearrangements in species of the remaining 7 families were also associated with some protein-coding genes.（7）Phylogenetic trees constructed based on both datasets（PCGs+r RNAs and PCGs）showed that Gamasina were monophyletic groups,and the species within families always clustered together preferentially with a high confidence level.Maximum likelihood trees and Bayesian trees constructed based on PCGs+r RNAs and Bayesian trees constructed from PCGs showed that the family Haemogamasidae was most closely related to the family Dermanyssidae.However,the maximum likelihood tree constructed based on PCGs showed that the family Haemogamasidae is a monophyletic group.The phylogenetic tree constructed provides strong evidence that the family Haemogamasidae does not belong to a subfamily of the family Laelapidae.Divergent times indicate that Gamasina originated during the Carboniferous period（ca.344.15 Mya）,with most species occurring during the Jurassic and Cretaceous periods.Conclusion:Phylogenetic analyses support that Gamasina is a monophyletic group and that the family Haemogamasidae does not belong to the subfamily Laelapidae.The divergent timing suggests that Gamasina originated in the Carboniferous period（ca.344.15 Mya）.Among the Gamasina,the mitochondrial genomes of families Parasitidae and Diplogyniidae were structurally stable,indicating that the mitochondrial genomes of families Parasitidae and Diplogyniidae were strongly conserved.Other 9 families,15 genera and 20 species showed different degrees of rearrangement of mitochondrial genomes,indicating that the arrangement pattern of Gamasina mitochondrial genomes was more variable at lower taxonomic orders.The results of this study have important theoretical implications for gaining insight into the evolutionary relationships of Gamasina and for advancing research in mitochondrial genomics and vector mite related fields in China. |
