Phylogenetic Analysis Of Zingiber Plants Based On Chloroplast Genome And ITS Sequences

The genus Zingiber of the Zingiberaceae is a large genus with rich diversity,distributed in tropical,subtropical,and in Far East Asia.This genus contains about100–150 species,with many species valued as important agricultural,medicinal and horticultural resources.According to the classical morphological classification method,Zingiber plants can be divided into three groups: sect.Zingiber,sect.Pleuranthesis and sect.Cryptanthium.In previous reserach,the phylogenetic relationships of Zingiber plants have been lacking in systematic studies due to insufficient phylogenetic information contained in the molecular markers used or sampling limitations.Based on the principle of representing the diversity of classification levels under the genus,this study used the chloroplast genome data of 20 Zingiber species and 74 nuclear gene ITS sequences to analyze the structural variation characteristics and evolutionary patterns of the chloroplast genome and ITS sequences of Zingiber using the maximum likelihood method(ML)and Bayesian inference(BI),respectively,to reconstruct the maternally inherited chloroplast genome tree and the biparentally inherited nuclear gene phylogenetic tree to reveal the phylogenetic relationships among ginger species from different genetic perspectives.The main results obtained are as follows:(1)The chloroplast genome of Zingiber is relatively conserved,and 8hypervariable regions were identified.The chloroplast genome of Zingiber plants is relatively conservative.The chloroplast genome(size range 162,507-163,711 bp)possess typical quadripartite structures that consist of a large single copy(LSC,86,986-88,200 bp),a small single copy(SSC,15,498-15,891 bp)and a pair of inverted repeats(IRs,29,765-29,934 bp).The genome structures,gene contents,amino acid frequencies and codon usage patterns are conservative in the genomes of Zingiber.The contraction and expansion of the reverse repeat region are not obvious.A total of eight highly variable regions,including seven intergenic regions(pet A-pab J,rbc L-acc D,rpl32-trn L,rps16-trn Q,trn C-psb M,psb C-trn S and ndh F-rpl32)and one gene region(ycf1),were identified in the chloroplast genome of Zingiber plants,can be as potential molecular markers for subsequent species identification studies.The analysis of sequence divergence indicates that the following genes undergo positive selection(ccs A,ndh A,ndh B,pet D,psb A,psb B,psb C,rbc L,rpl12,rpl20,rpl23,rpl33,rpo C2,rps7,rps12 and ycf3).These genes may play an important role in the adaptive evolution of Zingiber plants.(2)The phylogenetic relationship of Zingiber plants forms a nuclear-cytoplasmic conflict relationship.The chloroplast genome data of 14 newly sequenced Zingiber species(contains seven species of the genus Zingiber that are endemic to China)are presented for the first time,and the phylogenetic tree construction was performed jointly with the chloroplast data of 6 published species,for a total of 20 Zingiber species,and the results show that: twenty species of Zingiber plants are divided into three groups:sect.Zingiber,sect.Pleuranthesis,and sect.Cryptanthium.Sect.Pleuranthesis is located at the base of the phylogenetic tree and contains only one species,Z.ellipticum,which forms a sister group with the members of the remaining two groups.It is noteworthy that our results strongly support the sister relationship between sect.Zingiber and sect.Cryptanthium,which is not consistent with the grouping results of Theerakulpisut(2012),who concluded that sect.Zingiber and sect.Pleuranthesis form a sister relationship based on ITS sequence data.This phenomenon of nuclear-mass conflict needs further in-depth study.(3)The ITS variation sites of Zingiber plants were abundant,but the genetic distance between species was small,only 0.089.The polymorphism of ITS variation sites in Zingiber plants is insufficient,and the phylogenetic relationship among some Zingiber species still needs further study.Structural characterization of the ITS sequences of 74 Zingiber species showed that the ITS sequences of Zingiber plants were rich in variant loci,accounting for 46.63% of the total loci number;The genetic distance between Zingiber species is small,with an average genetic distance of 0.089;The phylogenetic tree divides Zingiber plants into four genealogical branches,supporting that Z.engganoense forms Branch I alone,which is located at the base of the phylogenetic tree,and Branches III and IV form sister groups with Branch II.In this study,we conducted a phylogenetic study of Zingiber based on genome resequencing data,analyzed the structural variation characteristics and evolutionary patterns of chloroplast genome and nuclear gene ITS sequences,reconstructed the molecular phylogenetic tree,which provides a solid foundation for the subsequent in-depth elaboration of phylogenetic relationships,medicinal resource development and germplasm resource identification of Zingiber plants.