Ferns, 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. With 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. Our 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.

Ferns and lycophytes have remarkably large genomes. However, little is known about how their genome size evolved in fern lineages. To explore the origins and evolution of chromosome numbers and genome size in ferns, we used flow cytometry to measure the genomes of 240 species (255 samples) of extant ferns and lycophytes comprising 27 families and 72 genera, of which 228 species (242 samples) represent new reports. We analyzed correlations among genome size, spore size, chromosomal features, phylogeny, and habitat type preference within a phylogenetic framework. We also applied ANOVA and multinomial logistic regression analysis to preference of habitat type and genome size. Using the phylogeny, we conducted ancestral character reconstruction for habitat types and tested whether genome size changes simultaneously with shifts in habitat preference. We found that 2C values had weak phylogenetic signal, whereas the base number of chromosomes (x) had a strong phylogenetic signal. Furthermore, our analyses revealed a positive correlation between genome size and chromosome traits, indicating that the base number of chromosomes (x), chromosome size, and polyploidization may be primary contributors to genome expansion in ferns and lycophytes. Genome sizes in different habitat types varied significantly and were significantly correlated with habitat types; specifically, multinomial logistic regression indicated that species with larger 2C values were more likely to be epiphytes. Terrestrial habitat is inferred to be ancestral for both extant ferns and lycophytes, whereas transitions to other habitat types occurred as the major clades emerged. Shifts in habitat types appear be followed by periods of genomic stability. Based on these results, we inferred that habitat type changes and multiple whole-genome duplications have contributed to the formation of large genomes of ferns and their allies during their evolutionary history.

Whole-genome duplication has been recognized as a major process in speciation of land plants, especially in ferns. Whereas genome downsizing contributes greatly to the post-genome shock responses of polyploid flowering plants, diploidization of polyploid ferns diverges by maintaining most of the duplicated DNA and is thus expected to be dominated by genic processes. As a consequence, fern genomes provide excellent opportunities to study ecological speciation enforced by expansion of protein families via polyploidy. To test the key predictions of this hypothesis, we reported the de novo genome sequence of Adiantum nelumboides, a tetraploid homosporous fern. The obtained draft genome had a size of 6.27 Gb assembled into 11,767 scaffolds with the contig N50 of 1.37 Mb. Repetitive DNA sequences contributed with about 81.7%, a remarkably high proportion of the genome. With 69,568, the number of predicted protein-coding genes exceeded those reported in most other land plant genomes. Intragenomic synteny analyses recovered 443 blocks with the average block size of 1.29 Mb and the average gene content of 16 genes. The results are consistent with the hypothesis of high ancestral chromosome number, lack of substantial genome downsizing, and dominance of genic diploidization. As expected in the calciphilous plants, a notable number of detected genes were involved in calcium uptake and transport. In summary, the genome sequence of a tetraploid homosporous fern not only provides access to a genomic resource of a derived fern, but also supports the hypothesis of maintenance of high chromosome numbers and duplicated DNA in young polyploid ferns.

Cryptic species comprise two or more taxa that are grounded under a single name because they are more-or-less indistinguishable morphologically. These species are potentially important for detailed assessments of biodiversity, but there now appear to be many more cryptic species than previously estimated. One taxonomic group likely to contain many cryptic species is Dicranopteris, a genus of forked ferns that occurs commonly along roadsides in Asia. The genus has a complex taxonomical history, and D. linearis has been particularly challenging with many intra-specific taxa dubiously erected to accommodate morphological variation that lacks clear discontinuities. To resolve species boundaries within Dicranopteris, we applied a molecular phylogenetic approach as complementary to morphology. Specifically, we used five chloroplast gene regions (rbcL, atpB, rps4, matK, and trnL-trnF) to generate a well-resolved phylogeny based on 37 samples representing 13 taxa of Dicranopteris, spanning the major distributional area in Asia. The results showed that Dicranopteris consists of ten highly supported clades, and D. linearis is polyphyletic, suggesting cryptic diversity within the species. Further through morphological comparison, we certainly erected Dicranopteris austrosinensis Y.H. Yan & Z.Y. Wei sp. nov. and Dicranopteris baliensis Y.H. Yan & Z.Y. Wei sp. nov. as distinct species and proposed five new combinations. We also inferred that the extant diversity of the genus Dicranopteris may result from relatively recent diversification in the Miocene based on divergence time dating. Overall, our study not only provided additional insights on the Gleicheniaceae tree of life, but also served as a case of integrating molecular and morphological approaches to elucidate cryptic diversity in taxonomically difficult groups.

Whole-genome duplications (WGDs) are widespread in plants and frequently coincide with global climatic change events, such as the Cretaceous–Tertiary (KT) extinction event approximately 65 million years ago (mya). Ferns have larger genomes and higher chromosome numbers than seed plants, which likely resulted from multiple rounds of polyploidy. Here, we use diploid and triploid material from a model fern species, Ceratopteris thalictroides, for the detection of WGDs. High-quality RNA-seq data was used to infer the number of synonymous substitutions per synonymous site (Ks) between paralogs; Ks age distribution and absolute dating approach were used to determine the age of WGD events. Evidence of an ancient WGD event with a Ks peak value of approximately 1.2 was obtained for both samples; however, the Ks frequency distributions varied significantly. Importantly, we dated the WGD event at 51–53 mya, which coincides with the Paleocene-Eocene Thermal Maximum (PETM), when the Earth became warmer and wetter than any other period during the Cenozoic. Duplicate genes were preferentially retained for specific functions, such as environment response, further support that the duplicates may have promoted quick adaption to environmental changes and potentially resulted in evolutionary success, especially for pantropical species, such as C. thalictroides, which exhibits higher temperature tolerance.