| Polypod ferns contain about 82% species diversity of extant ferns(8,700/10,600),which represent the second most species-rich lineage of vascular plants,only exceeded by angiosperms.The deep relationships of Polypodiales have been intensively investigated in the last 20 years;however,they remain as a highly dynamic research area in fern systematics.It was generally believed that polypod ferns diversified in the shadow of angiosperms in the Late Cretaceous.This hypothesis has been adopted widely since it was proposed,while it has been questioned by other some studies.However,previous time-estimates were based either on incomplete sampling or fossil recognition.We used a phylogenomic approach to resolve the relationships among the backbones of Polypodiales,and to date deepest divergences in polypod ferns with comprehensive taxon sampling,together with a good consideration of important fossil taxa,especially some well-preserved Early Cretaceous polypod fern fossils.We sampled 208 plastomes representing 199 species of 109 genera of all 28 polypod fern families,with 164 plastomes representing 160 species of 100 genera of 27 families newly generated in the present study.We also compared the diversification patterns of polypod ferns with those of angiosperms.The results showed that the plastid genomes of polypod ferns exhibited high stability in structure and gene content,and the variation in genomic structure and the changes in gene content were scattered among distant clades or individuals.Prominent features including RNA editing,IR expansion or contraction,and long noncoding sequence insertion were detected in the Polypodiales plastid genomes.The phylogeny based on 88 coding genes of 214 samples and eight analytical schemes recovered six main lineages of Polypodiales recognized as suborders;relationships at and above the familial level were largely identical.The suborders Saccolomatineae and Lindsaenieae were sister clades and in turn formed together(the Sa Cy Lo Li clade)the sister clade to all other Polypodiales.The Dennstaedtiineae and Pteridineae were sister clades that were together sister to a clade comprising Aspleniineae(eupolypods II)and Polypodiineae(eupolypods I).The relationships among Dennstaedtiineae,Pteridineae,and eupolypods still need further investigation because of the inconsistencies between the analyses of plastid DNA and AA matrices,and between the analysis of plastid and trascriptome data.Hypodematiaceae were the first diverging clade followed by Didymochlaenaceae in the Polypodiineae clade.Cystopteridaceae were sister to the remaining Aspleniineae that comprises two clades,namely the WOBAT clade(including Athyriaceae,Blechnaceae,Onocleaceae,Thelypteridaceae and Woodsiaceae)and the RHADD clade(including Aspleniaceae,Desmophlebiaceae,Diplaziopsidaceae,Hemidictyaceae and Rhachidosoraceae)in the Aspleniineae clade.Within the RHADD clade,Rhachidosoraceae were sister to the rest families,and Hemidictyaceae sister to Desmophlebiaceae.We estimated the divergence times of polypod ferns using both Penalized likelihood and Bayesian methods based on our phylogenetic framework,and relaxed molecular clocks incorporated 14 calibrations of 16 fossil record.Despite some substantial differences in the divergence ages estimated by ten analytical schemes with the combination of dating methods,root age constraints,and the placement of Athyrium-like fossils constraints,the results converged to a rather informative pattern.It was revealed that the stem of the Polypodiales probably diverged in the Triassic with only two out of ten calibration schemes suggested an origin in the late Permian.Similarly,the crown of the Polypodiales diverged in the Triassic or Early Jurassic.The early diversifications of Polypodiales can be divided into two stages.The initial stage occurred during the Triassic–Jurassic period,giving rise to the Saccolomatineae,Lindsaeineae,Dennstaedtiineae,Pteridineae,and eupolypods.The major stage occurred mainly during the Cretaceous,resulting in the radiation of Dennstaedtiineae,Pteridineae,and especially eupolypods.The stem eupolypods diverged in most schemes in the Early to Mid-Jurassic with only two schemes suggested a Triassic origin.The crown eupolypods diverged either in the Early Cretaceous or in the Late Jurassic.The vast majority of eupolypod families were estimated to originate in the Early Cretaceous and diverge in the Late Cretaceous to Paleogene.The density plots of the divergence time estimates for Polypodiales and angiosperms suggested that both angiosperms and polypod ferns diversified during the Cretaceous Terrestrial Revolution although both lineages show a cryptic pre-Cretaceous history.However,such a pre-Cretaceous history has been detected for neither angiosperms nor ferns in the fossil record until now.The initial establishment of the crown polypod ferns was date back to the Triassic,whereas the divergence of the main lineages of polypod ferns may reach their peak around 160–96 million years before the present.The largescale climate change from arid to temperate dominance during the Jurassic and the Early Cretaceous that driven by the breakup of Pangea,may have triggered the diversification of major lineages within both angiosperms and polypod ferns.More information about the fossil record,increased sampling,and arguably improved methodology are required to explain the evolutionary processes shaping the success of these lineages and in turn resulted in the dismiss of lineages that were dominant during most of the Mesozoic.Additionally,we tested the effect of plastid RNA editing on phylogenetic reconstruction using the most RNA-edited lineage in land plants,the spike mosses Selaginella as a model.The contrast phylogenetic analyses showed that there were substantial differences(e.g.,the placement of clade B)between the original and corrected datasets.Our empirical study provided evidence that the extreme C-to-U RNA editing in the coding regions of organellar genomes definitely altered the sequences used for phylogenetic reconstruction,and might even mislead the result of phylogenetic reconstruction. |
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