BackgroundFerns, originated about 360 million years ago, are the sister group of seed plants. Despite the remarkable progress in our understanding of fern phylogeny, with conflicting molecular evidence and different morphological interpretations, relationships among major fern lineages remain controversial.ResultsWith the aim to obtain a robust fern phylogeny, we carried out a large-scale phylogenomic analysis using high-quality transcriptome sequencing data, which covered 69 fern species from 38 families and 11 orders. Both coalescent-based and concatenation-based methods were applied to both nucleotide and amino acid sequences in species tree estimation. The resulting topologies are largely congruent with each other, except for the placement of Angiopteris fokiensis, Cheiropleuria bicuspis, Diplaziopsis brunoniana, Matteuccia struthiopteris, Elaphoglossum mcclurei, and Tectaria subpedata.ConclusionsOur result confirmed that Equisetales is sister to the rest of ferns, and Dennstaedtiaceae is sister to eupolypods. Moreover, our result strongly supported some relationships different from the current view of fern phylogeny, including that Marattiaceae may be sister to the monophyletic clade of Psilotaceae and Ophioglossaceae; that Gleicheniaceae and Hymenophyllaceae form a monophyletic clade sister to Dipteridaceae; and that Aspleniaceae is sister to the rest of the groups in eupolypods II. These results were interpreted with morphological traits, especially sporangia characters, and a new evolutionary route of sporangial annulus in ferns was suggested. This backbone phylogeny in ferns sets a foundation for further studies in biology and evolution in ferns, and therefore in plants.

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The eupolypods II ferns represent a classic case of evolutionary radiation and, simultaneously, exhibit high substitution rate heterogeneity. These factors have been proposed to contribute to the contentious resolutions among clades within this fern group in multilocus phylogenetic studies. We investigated the deep phylogenetic relationships of eupolypod II ferns by sampling all major families and using 40 plastid genomes, or plastomes, of which 33 were newly sequenced with next-generation sequencing technology. We performed model-based analyses to evaluate the diversity of molecular evolutionary rates for these ferns. Our plastome data, with more than 26,000 informative characters, yielded good resolution for deep relationships within eupolypods II and unambiguously clarified the position of Rhachidosoraceae and the monophyly of Athyriaceae. Results of rate heterogeneity analysis revealed approximately 33 significant rate shifts in eupolypod II ferns, with the most heterogeneous rates (both accelerations and decelerations) occurring in two phylogenetically difficult lineages, that is, the Rhachidosoraceae–Aspleniaceae and Athyriaceae clades. These observations support the hypothesis that rate heterogeneity has previously constrained the deep phylogenetic resolution in eupolypods II. According to the plastome data, we propose that 14 chloroplast markers are particularly phylogenetically informative for eupolypods II both at the familial and generic levels. Our study demonstrates the power of a character-rich plastome data set and high-throughput sequencing for resolving the recalcitrant lineages, which have undergone rapid evolutionary radiation and dramatic changes in substitution rates.

Protein-coding gene annotation. To search for homologous genes, the protein sequences from all ferns and lycophytes transcriptomes in the OneKP project 1 were retrieved and aligned to the A. capillus-veneris genome, using GeneWise 2 . For transcriptome-based prediction, nineteen transcriptomes covering the entire life cycle of A. capillus-veneris were generated in this study (Supplementary Table 8). RNA was extracted using the Qiagen RNeasy protocol and sequenced on an Illumina HiSeq 4000 with a 300 bp insert size. For transcriptome-based prediction, the HISAT2 3 and StringTie 4 programs were used for transcript assembly 5 . The program PASA (http://pasapipeline.github.io) was used to align spliced transcripts and annotate candidate genes. Ab initio prediction was performed with AUGUSTUS 6 , GlimmerHMM 7 , and SNAP 8 . Finally, nonredundant gene models were obtained with EVidenceModeler (version 1.1.0) 9 to integrate the gene models developed by different datasets.To validate the assembly quality, RNA-seq reads from nineteen tissues (Supplementary Table 8), together with publicly available EST sequences from the NCBI database (downloaded from http://togodb.dbcls.jp/library), were mapped to the A. capillus-veneris genome using HISAT2 3 and BLAT 10 with default parameters, respectively. The BLAT results were filtered with an identity and coverage cutoff of 0.9.Identification of noncoding RNAs. We used tRNAscan-SE (version 2.0rc2) 11 , with default parameters, to search for tRNAs in the A. capillus-veneris genome. A total of 1,624 tRNAs were found. Moreover, the Rfam14.0 database 12 , including 3,445 noncoding RNA families, was used to annotate additional noncoding RNAs (ncRNAs), including miRNAs, snRNAs, and tRNAs, using INFERNAL (version 1.1.2) 13 program.We predicted rRNA (5S, 5.8S, 28S, 18S) by using HMM searching based rRNA predicator Barrnap (version 0.9, https://github.com/tseemann/barrnap#barrnap), with default parameters. We finally identified 145 5S, 75 5.8S, 155 28S, and 165 18S sequences and their locations within the genome assembly of A. capillus-veneris.

The relationship of Hypolepis brooksiae, H. nigrescens, and H. scabristipes to the remainder of Hypolepis (Dennstaedtiaceae) has been questioned by previous authors based on their unique combination of morphological characters and different base chromosome number. Using four chloroplast genes including rbcL, atpA, rpL6, and rps4-trnS intergenic spacer (IGS) from 32 samples, representing 24 species of Dennstaedtiaceae, we recovered a clade comprising H. brooksiae and H. nigrescens, distinct from the remaining species of Hypolepis. This clade is resolved as sister to the clade comprising Blotiella, Paesia and Histiopteris. We reconstructed ancestral states of 16 morphological characters and found that this clade is distinguished by indeterminate, scandent leaves exhibiting rhythmic growth, provided with recurved black-tipped prickles, and stipule-like pinnules that protect the emerging crosier and pinnae departures, rachis-costa architecture where the adaxial sulcus is confluent with the next lower order, and a base chromosome number of x = 29. In light of this molecular and morphological evidence, we describe a new genus, Hiya, and provide nomenclatural combinations to accommodate the three known species segregated from Hypolepis: Hiya brooksiae, Hiya nigrescens, and Hiya scabristipes.