Comparative Analysis Of Mitochondrial Genome Of Macrophthalmidae Species And Phylogenetic Study Of Brachyura

Brachyura(Decapoda)are true crabs,which are widely found in oceans,fresh water,and even land around the world.For a long time,the species classification and phylogenetic relationships of Brachyura are still controversial issues because of their similarity in morphology.Big-eyed crabs(Ocypodoidea,Macrophthalmidae)play an important role in marine ecosystems.However,the systematic studies of Macrophthalmidae species started late both at home and abroad,and most of the studies focused on morphological classification and ecological habits.Up to now,there has still been a lack of comprehensive systematic studies on the phylogeny of Macrophthalmidae species.In recent years,mitochondrial genomes(including sequence and gene arrangement)have been widely used in taxonomic identification and phylogenetic evolution,and solving many controversial taxonomic and phylogenetic problems.In this study,three species of including Venitus latreillei,Macrophthalmus japonicus,and M.abbreviatus were selected as Macrophthalmidae representatives.The complete mitochondrial genome(mitogenome)sequence was determined and assembled.The mitogenomes of the three species were 15,747,16,170 and 16,322 bp in length;respectively,all containing 37 genes and a long control region(CR).There were similar overlapping regions and spacer regions between the genes,and all showed obvious AT bias in base composition.Most genes use ATN and TAN as start and stop codons;respectively.A few genes use GTG and GAA as start and stop codons;respectively.The frequency of codon use of 13 protein-coding genes showed a strong bias,and the third sites A and T had the highest frequency of codon used.Comparative analysis of mitogenomes of six Macrophthalmidae species showed that the length variation of mitochondrial genome of this Family was mainly due to the variation of control region,and the variation of control region length was mainly due to the difference of tandem repeats.The longest tandem unit was 309 bp with 3.6 repetitions(M.pacificus),while the shortest tandem unit was 2 bp with 46 repetitions(V.latreillei),52.5 repetitions(Euplax sp.)and 18.5 repetitions(M.pacificus).Of course,there are no tandem repeats in the control regions of M.japonicus and M.darwinensis.Protein coding gene selection pressure analysis showed that all mitochondrial protein-coding genes of Macrophthalmidae were less than 1,indicating that they had not been subjected to natural selection in the evolutionary process.The ω values of protein-coding genes involved in the synthesis of respiratory chain complex I subunit(ND1-ND6 and ND4L)were relatively high on the whole.The ω values of the genes involved in complex Ⅲ subunit synthesis(Cyt b)and complex Ⅳ subunit synthesis(COⅠCOⅢ)were relatively low.Gene order analysis showed that all Macrophthalmidae species experienced the same gene rearrangement events,which could be explained by the duplication-random loss model.Finally,the phylogenetic relationships of Brachyura were reconstructed based on 13 mitochondrial protein-coding genes.The results showed that in the Family level,Homolidae,Raninidae,Xanthidae and Majidae were non-monophyletic groups,while the rest were monophyletic groups.However,it is worth noting that the non-monophyletic groups of these four families are caused by the fact that the species clusing with them are all single representative of the family.Therefore,morphological and molecular biological studies on more representative species are needed in the future to confirm whether they are really nonmonophyletic groups.In the Superfamily level,the paraphyly of the two superfamilies has also been verified,which is consistent with most studies,but the relationship between the two families and their internal species is still controversial.In the higher taxonomic level,Heterotremata are paraphyletic group,and Thoracotremata are monophyletic group,including Grapsoidea,Ocypodoidea,Cryptochiroidea and Pinnotheroidea.In the Suborder level,Brachyura is a monophyletic group and can be divided into two branches,one of which is Dromiacea located at the base of the phylogenetic tree,the other is composed of Eubrachyura and Rainoida.However,due to the limited samples and molecular data available,the evolutionary position of the two groups in Brachyura remains to be discussed.