| In this study,the species of Margattea were defined by morphological data and cox1 gene using the molecular species delimitation method--General Mixed Yule Coalescent(GMYC).Then,the mitochondrial genome data of 24 species of Pseudophyllodromiinae were sequenced.The characteristics of mitochondrial genome of 24 species of Pseudophyllodromiinae were compared and analyzed by means of comparative mitochondrial genome in order to infer the differences among the species.After that,two different data sets(PCG12r RNAt RNA and PCG12 r RNA)of mitochondrial genome were combined for phylogenetic analysis using maximum likelihood and Bayesian methods to explore the phylogenetic relationship and divergence time of the main clades of the subfamily Pseudophyllodromiinae.The following results were obtained:(1)77 cox1 sequences(68 by self-test and 9sequences downloaded on NCBI)of Margattea were analyzed by the maximum likelihood method and delimited species using GMYC.Compared with the result of morphological identification,there is a minor difference in these two methods.GMYC analysis resulted in 21 entities(confidence interval: 20–22).The MOTUs were in nearly perfect accord with assigned morphospecies(16).Although a large genetic distance existed(2.9% in M.concava,3.1% in M.speciosa and 5.9% in M.bisignata),M.speciosa,M.concava and M.bisignata were determined to intraspecific differences owing to the slight differences existed.Finally,7 new species of Margattea were defined according to molecular and morphological characteristics:Margattea deltodonta sp.nov.,Margattea cuspidata sp.nov.,Margattea caudata sp.nov.,Margattea paratransversa sp.nov.,Margattea disparilis sp.nov.,Margattea transversa sp.nov.,and Margattea bicruris sp.nov.DNA barcode technology can be used as a rapid and independent technique to help define the species of Margattea,and can also be used for matching male and female.(2)The comparative analysis results of 25 mitochondrial genome data(24self-tests plus one data from NCBI)are provided and compared.It is found that there is no rearrangement phenomenon in species of Pseudophyllodromiinae,and the differences between genera of Pseudophyllodromiinae cannot be clearly defined only according to the existing data.(3)The phylogenetic analysis shows that Pseudophyllodromiinae,and the genera Margattea,Balta(Chorisoserrata,Amazonina and Euphylodomia are only one species)are monophyletic.Among them,Margattea and Chorisoserrata are recovered to be the sister group to each other,(Margattea + Chorisoserrata)is the sister group of Amazonina.Allacta and Sorineuchora are recovered to be paraphyletic both in ML and BI analysis.A.australiensis and A.bimaculata are clustered together;A.hainanensis,A.bruna and A.robusta are clustered together.S.lativitrea and S.bimaculata are separately clustered.According to the phylogenetic results and morphological characteristics,two new genera are defined: Epiallacta gen.nov.and Episorineuchora gen.nov.We transfered A.australiensis and A.bimaculata to Epiallacta gen.nov.,and transfered S.lativitrea to Episorineuchora gen.nov.(4)Based on 13 protein genes(excluding the third codon)and 2 r RNA genes of the mitochondrial genome,9 fossil calibration were selected for the time divergence analysis of Pseudophyllodromiinae.The results showed that the divergence time between Pseudophyllodromiinae and its sister group was 169.26Ma(95% credibility interval: 139.36-197.77Ma).The first divergence event in this subfamily occurred at157.59Ma(130.40-185.16Ma).At 132.11Ma(108.22-156.48Ma),the Epiallacta and its sister group had a divergence event.The time of divergence event between Allacta and its sister group was 107.64 Ma(86.67-129.81 Ma).The occurrence of divergence between Sorineuchora and Episorineuchora was 102.71 Ma(78.99-125.91 Ma CI).The time of divergence event between Margattea and its sister group was110.41Ma(89.94-133.36Ma),while Balta diverged from its sister group at 86.27Ma(69.19-104.77Ma). |
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