| Zygophyllaceae is an old family with about 285 species.As the largest genus in Zygophyllaceae/this family,Zygophyllum L.consists of about 150 species in the worldwide and more than 20 species in China.This genus mainly distributes in the arid and semi-arid areas of northwest China and is an important component of the ecosystem in this area.Most species in Zygophyllum are herb types and only two species(Z.xanthoxylon and Z.kaschgaricum)are shrub types in China,which are aways excellent resistance plants,medicinal plants and have important ecological value.In the fragile ecosystem of the northwest China,Z.xanthoxylon is a dominant species and keeps the ecological equilibrium in these communities.Up to now,some species in Zygophyllum has been controversial due to lacking of detailed description of morphological characteristics and deep exploration of infragenric relationship within Zygophyllum in previous studies.In this study,we observed the morphology or micromorphology of leaf epidermises,flowers,fruits and seeds of Zygophyllum using stereo microscope and scanning electron microscope(SEM).Based on chloroplast genome sequences generated by high-throughput sequencing and SNP developed via specific-locus amplified fragment sequencing(SLAF-seq)technique,we reconstructed phylogenetic relationship,estimated the divergence time and inferred the character evolution of Zygophyllum.At the same time,polymorphic EST-SSR loci were used for population genetic analysis Z.xanthoxylon.The major results are showed as follows.(1)We observed and described the morphology of the leaf epidermis for 14 species,flowers of 14 species,morphology and micromorphology of seed for 12 species in Zygophyllum.Leaf epidermis of 14 species had thick cuticle with granulatus and lamellate wax.Stomata were distributed in the adaxial and abaxial epidermis of leaves,and most of stomata were always sunken.There are granulatus and lamellate wax on the outer and inner margin shape of outer stomatal.The inner margin shape of outer stomatal is mostly sinuolate.Petals are longer or slightly shorter than sepals,and appendages are one third or less than half as long as filaments with often transparent,rarely orange red.Appendages surface are glabrous,rarely nipple-like,which apex is sinuolate,dentate or denticulate.The 12 species had baculiform raise,rarely clusters of trichome on the surface of dry seed,but the amount of transparent mucilaginous on wet seed.Under the suitable light,the seeds of Zygophyllum species were transmittance except Z.potaninii and Z.sinkiangense.The morphological and micromorphological features of Z.xanthoxylon and Z.kaschgaricum are highly similar,and their fruit morphology differences are intraspecific variation.(2)In this study,we sampled 24 individuals of Zygophyllum representing 15 species and obtained their complete chloroplast(cp)genomes.For comparison,we also sequenced chloroplast genomes of two species of Peganum from China representing the closely allied Nitrariaceae family.The 24 cp genomes of Zygophyllum were smaller and ranged in size from 104,221 to 106,286 bp with classical four-phase structure.These cp genomes contained111-112 genes,including 74-75 protein-coding genes(PCGs),four r RNA genes,and 33 transfer RNA genes,and all cp genomes showed similar gene order,content,and structure.The cp genomes of Zygophyllum appeared to lose some genes including ndh genes and r RNA genes,which four r RNA genes were in the SSC region,not the IR regions.However,the SC and IR regions had greater similarity within Zygophyllum than between this genus and Peganum.We detected nine highly variable intergenic spacers: mat K-trn Q,psa C-rps15,psb Z-trn G,rps7-trn L,rps15-trn N,trn E-trn T,trn L-rpl32,trn Q-psb K and trn S-trn G.Additionally,we identified 2,459 simple sequence repeats(cp SSR)markers shared among the genomes of the 24 Zygophyllum samples and seven cp SSRs that were unique to the species of Zygophyllum.(3)We obtained 18,327 single nucleotide polymorphism(SNP)from 55 individuals representing 15 Zygophyllum species and three individuals of Tetraena mongolica(Zygophylloideae)using the specific-locus amplified fragment sequencing(SLAF-seq)technique.Using the nuclear and plastome datasets,a well-resolved phylogeny of Zygophyllum in China strongly supported the division of Chinese Zygophyllum into herbaceous and woody clades,and the herbaceous clade comprised a basal grade and three subclades.The positions of several species changed in the phylogenetic trees,and there was different in the basal grade of the herbaceous clade.The fruit characteristics,including shape,winged or not,dehiscent or indehiscent,supported the phylogenetic relationship of herbaceous branches.Based on the morphological and molecular evident,we considered that Z.kaschgaricum was merged into Z.xanthoxylon,while treated Sarcozygium as a subgenus of Zygophyllum.We confirmed most Zygophyllum species,such as Z.fabago,not Z.brachypterum,but the phylogenetic relationship of species within branch of Z.rosowii needed further study.In addition,the divergence time of China Zygophyllum was in the early Miocene,and the divergence time of herbaceous clade was earlier than the woody clade.The ancestral states reconstruction of the four morphology characteristics showed fruit indehiscence type,fruit wingless type,2 leaflets type and 5 carpel type were the ancestral states in Chinese Zygophyllum.(4)Based on transcriptomic sequence of Z.xanthoxylon,we developed polymorphic EST-SSR loci and identified 22 pairs primer as highly polymorphic used for population genetic analysis.The genetic diversity of Z.xanthoxylon gradually decreased from east to west.The UPGMA tree showed that Z.kaschgaricum individuals with small fruit and nearly narrow ovate did not form an independent clade and nested with Z.xanthoxylon individuals.In addition,the UPGMA analyses indicated apparent geographical structure among populations and geographically close individuals clustered.AMOVA analysis showed that32.62% genetic variation was among the populations,indicating a high level of genetic differentiation among the overall populations and a high genetic variation(67.18%)within populations.Geographical distance significantly affected genetic differentiation.Few gene flow among populations can not resist genetic drift,which was certain related to the existence of natural barriers,such as several deserts and mountains.The 29 populations were divided into east lineage and west lineage,in which 5 populations located in the dry valley of Tianshan Mountain showed apparent gene flow,and we inferred Z.xanthoxylon populations in the westernmost point might be a cryptic species.Z.kaschgaricum samples did not formed an independent clade in the UPGMA tree and independent lineage,hence we suggested Z.kaschgaricum merging into Z.xanthoxylon. |
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