Development And Application Of Single Copy Molecular Markers In Cyatheaceae Based On Transcriptomics

Cyatheaceae is a very unique natural group of ferns.Most of the species in this family are large tree-like ferns,which are also known as scaly tree ferns due to the large number of scales on their stems and leaves.The origin of Cyatheaceae can date back to late-Cretaceous.The phylogeny of Cyatheaceae remains unresolved for multiple resons.With the development of modern molecular biology,transcriptome sequencing may provide new evidence for the phylogenetic study of Cyatheaceae.Single-copy nuclear genes have many characteristics conducive to phylogenetic analysis.As a powerful and cost-efficient tool,transcriptome sequencing technology has been increasingly used to screen a large number of single-copy nuclear gene markers.The purpose of this study is to identify high resolution single-copy nuclear loci on phylogenetic analysis of Cyatheaceae at the species and genus level,and to provide new data for the classification of Cyatheaceae.In this study,we generated five transcriptomes of Cyatheaceae and used them to search for single-copy nuclear loci for phylogenetic reconstruction.We identified a total of 818 candidate single-copy loci across multiple Cyatheaceae species.Using these single-copy loci data,we designed 58 pairs of primers,and from which we screened 12 pairs with high amplification and sequencing success rate.Further,we have taken a large scale of sampling of all 15 scaly tree ferns known in China and Vietnam,including 96 groups and 543 individuals.Then,we selected 76 representative individuals for DNA extraction,and amplified with 12 pairs primers.Finally,we conducted a series of phylogenetic analysis on the obtained sequence data.The results are as follow:(1)The concatenated alignment was 8,321 bp long,of which 7,562 characters were constant,163 were parsimony-uninformative,and 596 were parsimony-informative.(2)The phylogenetic analyses based on either multispecies coalescent or concatenation models results showed that all samples were clustered into three clades,i.e.Sphaeropteris clade,Alsophila clade and Gymnosphaera clade.(3)The Gymnosphaera clade were further resolved as four subclades: I,the samples of G.gigantea,G.glabra,G.andersonii,and G.khasyana;II,those of G.denticulata.and G.metteniana;III,all samples of G.podophylla and G.bachmaensis and the sample of the undescribed Gymnosphaera;and IV,all samples of G.salletii and G.austroyunnanensis(4)Based on the results from the BEAST,the crown age of Cyatheaceae was estimated at 89.2 Ma in the late-Cretaceous(95% HPD 58.1– 101.6 Ma),the AlsophilaGymnosphaera split at 60.2 Ma(around the Paleocene-Eocene boundary,95% HPD 42.2–71.8 Ma),the crown group age of the Gymnosphaera clade was estimated to be 11.8 Ma(middle Miocene,95% HPD 6.0–16.2 Ma),and the Alsophila clade had the crown group origins at late Miocene(6.7 Ma,95% HPD 2.8–10.8 Ma).