A Phylogenetic Study Of Anaphalis Based On Chloroplast Genomes

Anaphalis DC is a large genus of Gnaphalieae in the Asteraceae/Compositae family,with more than 110 species.It is mainly distributed in the tropical and subtropical regions of Asia,with East Asia as the center of distribution,extending to India,Indo-China Peninsula,tropics of Malaysia and a few in Europe and North America.There are many controversy in systematic relationship between Anaphalis species and related genus,due to its large number of species,variety of morphological traits,overlapping morphological characteristics,and widely distributed all over the world.Most Anaphalis species show polytype phenomenon in morphology.And hybridization and polyploidization are general in Anaphalis.Chloroplast genome have the characteristics of small genome,but contain a lot of genetic information;easily extracted and separated;and obvious differences at the molecular level,is widely used in the study of species phylogeny.In this study,61taxa of Anaphalis?belong to 47 species?and 3 Pseudognaphalium species were sequenced by high-throughput sequencing technique,and all chloroplast genome sequences were annotated by bioinformatics methods.The genomic sequences were compared and analyzed.The chloroplast genome sequences and nuclear gene sequences were used to study the phylogeny within Anaphalis and between Anaphalis and its related genera.And the phylogenetic relationships of Anaphalis were also elucidated by morphological characteristics.The main contents and results of this study are as follows:1.In this study,the complete chloroplast genome sequences of 53 taxa of Anaphalis and 3 Pseudognaphalium species were obtained by high-throughput sequencing technology,and we also got incomplete chloroplast genome sequences of8 Anaphalis taxa.According to the biological information analysis of 53 complete chloroplasts,the chloroplast genomes have a typical quadripartite structure,The total length is between 151082 and 153361 bp,among which,the length of the large single-copy?LSC?is 83306-85102 bp,the length of the small single-copy?SSC?is18046-18571 bp,the inverse repeat?IR?region 24831-24928 bp.A total of 132 genes?87 protein coding genes,37 tRNA genes,and 8 rRNA genes?were annotated.Among them,34 chloroplast sequences,such as A.aureopunctata,A.szechuanensis and A.bicolor,losted a tRNA gene,trnT-GGU,which contained a total of 131 genes.The overall GC content of Anaphalis was 37.1%and 37.2%,respectively.Using bioinformatics analysis,a total of 3670 SSR loci were detected in 53 chloroplast genomes of Anaphalis,of which mononucletoide repeats were the highest,following by tetranucleotide and trinucleotide repeats,and only a few species,such as A.busua and A.virgata,contain hexanucleotide repeats.2.The mVISTA was used to analyses the consistency of the chloroplast genome sequence.The chloroplast genome of Anaphalis was conservative,and nine regions with great variation were screened out,which contained abundant information sites.It can provide important information for the study of species identification and phylogeny.The IR region contraction and expansion of chloroplast genome were analyzed.The results showed that the boundary difference of IR region was highly conservative,and the size of chloroplast genome was not related to the contraction and expansion of IR region.It was mainly affected by the length of LSC region.3.Maximum likelihood?ML?and maximum parsimony?MP?,were used to construct phylogeny trees based on the chloroplast genome sequences and two nuclear gene sequences,respectively.In phylogenetic trees based on chloroplast genomes,Anaphalis was divided into two large branches.The first clade consisting of A.subdecurrens,A.margaritacea,A.royaleana,A.nubigna,A.hellwigii,A.contorta,A.racemifera,A.tenella,A.cavei,A.busua,A.nepalensis,A.acutifolia,A.triplinervis,A.virgata,A.roseo-alba and A.aurora(ML_BS=99%,MP_BS=100%),The second clade includes most of the remaining Anaphalis speices(ML_BS=100%,MP_BS=100%).Compared with the second clade,the chloroplast genomes of the first clade have trnT-GGU gene,6bp deletion in ycf3,6bp deletion in ycf2 and 9bp insertion in ndhF.According to the morphological characteristics analysis,the first clade showed that the micromorphological characteristics of achenes were reticulated transverse rod-shaped protuberance and honeycomb-shaped inflorescence receptacle,and leaf base attenuate.The trnT-GGU gene of chloroplast genome in the second clade species was lost,and the microsurface morphology of achenes was tongue-like protuberance,and the inflorescences had border-shaped protuberances,and leaf base decurrent.In the phylogenetic tree based on ITS and ETS,the species were also divided into two large branches,Among them,A.cavei,A.contorta,A.tenella,A.busua gathered in the first clade and formed a sister group with A.aureopunctata,A.sinica,A.contortiformis,etc.However,in the phylogenetic tree constructed by the chloroplast genome,A.cavei,A.contorta,A.tenella,A.busua are brought together(ML_BS=100%,MP_BS=100%)and associated with A.margaritacea,A.triplinervis,A.hellwigii et al.gather in the first clade.All Anaphalis species used were nested with Pseudognaphalium and Helichrysum in the two phylogenetic trees.So my results did not support that Anaphalis was a monophyletic group.Compared with the phylogenetic tree based on nrITS and nrETS,the phylogenetic tree based on chloroplast genome belongs to maternal heredity and is consistent with the morphological characteristics,which can reflect the phylogenetic relationship of Anaphalis more realistically than the phylogenetic tree based on nuclear gene sequences.However,in the evolution process,there are much phenomena,such as interspecific hybridization and polyploidy,which leads to confusion in the relationship between phylogenetic tree species based on nuclear gene sequences.4.in combination with the phylogenetic tree and morphological characters constructed from chloroplast genome,we suggest that Anaphalis should be divided into two subgenera,represented by A.margaritacea and A.sinica,respectively.Subgenus I includes species in the first clade,whose chloroplast genome contains the trnT-GGU gene,but compared with other Compositae,the gene has 9bp deletion and6bp insertion,and the surface of the achene is characterized by reticulate and transverse rod-shaped protuberances,receptacle honeycomb,leaf base attenuate;Subgenus II includes species in the second clade.the chloroplast genome of trnT-GGU gene is lost,and the surface of achene is characterized by tongue-shaped protuberance,floral bracelet with border-shaped protuberance,and the leaf base decurrent;.