Whole Genome Duplication Events Likely Contributed to the Aquatic Adaptive Evolution of Parkerioideae.

As the only aquatic lineage of Pteridaceae, Parkerioideae is distinct from many xeric-adapted species of the family and consists of the freshwater Ceratopteris species and the only mangrove ferns from the genus Acrostichum. Previous studies have shown that whole genome duplication (WGD) has occurred in Parkerioideae at least once and may have played a role in their adaptive evolution; however, more in-depth research regarding this is still required. In this study, comparative and evolutionary transcriptomics analyses were carried out to identify WGDs and explore their roles in the environmental adaptation of Parkerioideae. Three putative WGD events were identified within Parkerioideae, two of which were specific to Ceratopteris and Acrostichum, respectively. The functional enrichment analysis indicated that the lineage-specific WGD events have played a role in the adaptation of Parkerioideae to the low oxygen concentrations of aquatic habitats, as well as different aquatic environments of Ceratopteris and Acrostichum, such as the adaptation of Ceratopteris to reduced light levels and the adaptation of Acrostichum to high salinity. Positive selection analysis further provided evidence that the putative WGD events may have facilitated the adaptation of Parkerioideae to changes in habitat. Moreover, the gene family analysis indicated that the plasma membrane H+-ATPase (AHA), vacuolar H+-ATPase (VHA), and suppressor of K+ transport growth defect 1 (SKD1) may have been involved in the high salinity adaptation of Acrostichum. Our study provides new insights into the evolution and adaptations of Parkerioideae in different aquatic environments.

Geographical distribution and conservation strategy of national key protected wild plants of China.

National key protected wild plants (NKPWPs) are considered flagship species for plant diversity conservation in China. Using data for 1101 species, we characterized NKPWPs distribution patterns in China and assessed conservation effectiveness and conservation gaps. In total, 4880 grid cells at a 20 × 20 km resolution were filled with occurrence records for NKPWPs. We identified 444 hotspot grid cells and 27 diversity hotspot regions, containing 92.37% of NKPWPs. However, 43.24% of these hotspot grid cells were fully or partially covered by national nature reserves (NNRs), where 70.21% of species were distributed. Approximately 61.49% of the NKPWPs species were protected by NNRs, but the populations or habitats of 963 species were partially or fully outside of NNRs. With global warming, the overall change in the extent of suitable habitats for NKPWPs is expected to be small, however, habitat quality in some areas with a high habitat suitability index will decrease.

Insights into cryptic speciation of quillworts in China.

Cryptic species are commonly misidentified because of high morphological similarities to other species. One group of plants that may harbor large numbers of cryptic species is the quillworts (Isoëtes spp.), an ancient aquatic plant lineage. Although over 350 species of Isoëtes have been reported globally, only ten species have been recorded in China. The aim of this study is to better understand Isoëtes species diversity in China. For this purpose, we systematically explored the phylogeny and evolution of Isoëtes using complete chloroplast genome (plastome) data, spore morphology, chromosome number, genetic structure, and haplotypes of almost all Chinese Isoëtes populations. We identified three ploidy levels of Isoëtes in China-diploid (2n = 22), tetraploid (2n = 44), and hexaploid (2n = 66). We also found four megaspore and microspore ornamentation types in diploids, six in tetraploids, and three in hexaploids. Phylogenetic analyses confirmed that I. hypsophila as the ancestral group of the genus and revealed that Isoëtes diploids, tetraploids, and hexaploids do not form monophyletic clades. Most individual species possess a single genetic structure; however, several samples have conflicting positions on the phylogenetic tree based on SNPs and the tree based on plastome data. All 36 samples shared 22 haplotypes. Divergence time analysis showed that I. hypsophila diverged in the early Eocene (∼48.05 Ma), and most other Isoëtes species diverged 3-20 Ma. Additionally, different species of Isoëtes were found to inhabit different water systems and environments along the Yangtze River. These findings provide new insights into the relationships among Isoëtes species in China, where highly similar morphologic populations may harbor many cryptic species.

Untying the Gordian knot of plastid phylogenomic conflict: A case from ferns.

Phylogenomic studies based on plastid genome have resolved recalcitrant relationships among various plants, yet the phylogeny of Dennstaedtiaceae at the level of family and genera remains unresolved due to conflicting plastid genes, limited molecular data and incomplete taxon sampling of previous studies. The present study generated 30 new plastid genomes of Dennstaedtiaceae (9 genera, 29 species), which were combined with 42 publicly available plastid genomes (including 24 families, 27 genera, 42 species) to explore the evolution of Dennstaedtiaceae. In order to minimize the impact of systematic errors on the resolution of phylogenetic inference, we applied six strategies to generate 30 datasets based on CDS, intergenic spacers, and whole plastome, and two tree inference methods (maximum-likelihood, ML; and multispecies coalescent, MSC) to comprehensively analyze the plastome-scale data. Besides, the phylogenetic signal among all loci was quantified for controversial nodes using ML framework, and different topologies hypotheses among all datasets were tested. The species trees based on different datasets and methods revealed obvious conflicts at the base of the polypody ferns. The topology of the "CDS-codon-align-rm3" (CDS with the removal of the third codon) matrix was selected as the primary reference or summary tree. The final phylogenetic tree supported Dennstaedtiaceae as the sister group to eupolypods, and Dennstaedtioideae was divided into four clades with full support. This robust reconstructed phylogenetic backbone establishes a framework for future studies on Dennstaedtiaceae classification, evolution and diversification. The present study suggests considering plastid phylogenomic conflict when using plastid genomes. From our results, reducing saturated genes or sites can effectively mitigate tree conflicts for distantly related taxa. Moreover, phylogenetic trees based on amino acid sequences can be used as a comparison to verify the confidence of nucleotide-based trees.

The complete chloroplast genome sequence of the water fern Ceratopteris thalictroides (Pteridaceae).

This work determined and analyzed the complete chloroplast genome sequence of Ceratopteris thalictroides (Linnaeus) Brongniart 1822 (Pteridaceae). The results indicate that the total chloroplast genome size of C. thalictroides is 149,399 bp in length, and the genome contains a large single-copy (LSC) region of 83,580 bp, a small single-copy (SSC) region of 21,241 bp, and a pair of inverted repeat (IR) regions of 22,289 bp. The GC content of C. thalictroides is 36.7%. The genome encodes a total of 131 unique genes, including 82 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The phylogenetic analysis results strongly suggest that C. thalictroides is closely related to C. cornuta.

Genomic insights into genetic diploidization in the homosporous fern Adiantum nelumboides.

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.

Plastid Phylogenomics and Plastomic Diversity of the Extant Lycophytes.

Although extant lycophytes represent the most ancient surviving lineage of early vascular plants, their plastomic diversity has long been neglected. The ancient evolutionary history and distinct genetic diversity patterns of the three lycophyte families, each with its own characteristics, provide an ideal opportunity to investigate the interfamilial relationships of lycophytes and their associated patterns of evolution. To compensate for the lack of data on Lycopodiaceae, we sequenced and assembled 14 new plastid genomes (plastomes). Combined with other lycophyte plastomes available online, we reconstructed the phylogenetic relationships of the extant lycophytes based on 93 plastomes. We analyzed, traced, and compared the plastomic diversity and divergence of the three lycophyte families (Isoëtaceae, Lycopodiaceae, and Selaginellaceae) in terms of plastomic diversity by comparing their plastome sizes, GC contents, substitution rates, structural rearrangements, divergence times, ancestral states, RNA editings, and gene losses. Comparative analysis of plastid phylogenomics and plastomic diversity of three lycophyte families will set a foundation for further studies in biology and evolution in lycophytes and therefore in vascular plants.

Phylogenomic Analysis Reconstructed the Order Matoniales from Paleopolyploidy Veil.

Phylogenetic conflicts limit our understanding of the evolution of terrestrial life under multiple whole genome duplication events, and the phylogeny of early terrestrial plants remains full of controversy. Although much incongruence has been solved with so-called robust topology based on single or lower copy genes, the evolutionary mechanisms behind phylogenetic conflicts such as polyploidization remain poorly understood. Here, through decreasing the effects of polyploidization and increasing the samples of species, which represent all four orders and eight families that comprise early leptosporangiate ferns, we have reconstructed a robust phylogenetic tree and network with 1125 1-to-1 orthologs based on both coalescent and concatenation methods. Our data consistently suggest that Matoniales, as a monophyletic lineage including Matoniaceae and Dipteridaceae, should be redefined as an ordinal rank. Furthermore, we have identified and located at least 11 whole-genome duplication events within the evolutionary history of four leptosporangiates lineages, and associated polyploidization with higher speciation rates and mass extinction events. We hypothesize that paleopolyploidization may have enabled leptosporangiate ferns to survive during mass extinction events at the end Permian period and then flourish throughout the Mesozoic era, which is supported by extensive fossil records. Our results highlight how ancient polyploidy can result in rapid species radiation, thus causing phylogenetic conflicts yet allowing plants to survive and thrive during mass extinction events.

Lycophyte transcriptomes reveal two whole-genome duplications in Lycopodiaceae: Insights into the polyploidization of Phlegmariurus.

Lycophytes are an ancient clade of the non-flowering vascular plants with chromosome numbers that vary from tens to hundreds. They are an excellent study system for examining whole-genome duplications (WGDs), or polyploidization, in spore-dispersed vascular plants. However, a lack of genome sequence data limits the reliable detection of very ancient WGDs, small-scale duplications (SSDs), and recent WGDs. Here, we integrated phylogenomic analysis and the distribution of synonymous substitutions per synonymous sites (Ks) of the transcriptomes of 13 species of lycophytes to identify, locate, and date multiple WGDs in the lycophyte family Lycopodiaceae. Additionally, we examined the genus Phlegmariurus for signs of genetic discordance, which can provide valuable insight into the underlying causes of such conflict (e.g., hybridization, incomplete lineage sorting, or horizontal gene transfer).We found strong evidence that two WGD events occurred along the phylogenetic backbone of Lycopodiaceae, with one occurring in the common ancestor of extant Phlegmariurus (Lycopodiaceae) approximately 22-23 million years ago (Mya) and the other occurring in the common ancestor of Lycopodiaceae around 206-214 Mya. Interestingly, we found significant genetic discordance in the genus Phlegmariurus, indicating that the genus has a complex evolutionary history. This study provides molecular evidence for multiple WGDs in Lycopodiaceae and offers phylogenetic clues to the evolutionary history of Lycopodiaceae.

Corrigendum: Phylogeny and Taxonomy on Cryptic Species of Forked Ferns of Asia.

[This corrects the article DOI: 10.3389/fpls.2021.748562.].

Convergent molecular evolution of phosphoenolpyruvate carboxylase gene family in C4 and crassulacean acid metabolism plants.

Phosphoenolpyruvate carboxylase (PEPC), as the key enzyme in initial carbon fixation of C4and crassulacean acid mechanism (CAM) pathways, was thought to undergo convergent adaptive changes resulting in the convergent evolution of C4 and CAM photosynthesis in vascular plants. However, the integral evolutionary history and convergence of PEPC in plants remain poorly understood. In the present study, we identified the members of PEPC gene family across green plants with seventeen genomic datasets, found ten conserved motifs and modeled three-dimensional protein structures of 90 plant-type PEPC genes. After reconstructing PEPC gene family tree and reconciled with species tree, we found PEPC genes underwent 71 gene duplication events and 16 gene loss events, which might result from whole-genome duplication events in plants. Based on the phylogenetic tree of the PEPC gene family, we detected four convergent evolution sites of PEPC in C4 species but none in CAM species. The PEPC gene family was ubiquitous and highly conservative in green plants. After originating from gene duplication of ancestral C3-PEPC, C4-PEPC isoforms underwent convergent molecular substitution that might facilitate the convergent evolution of C4 photosynthesis in Angiosperms. However, there was no evidence for convergent molecular evolution of PEPC genes between CAM plants. Our findings help to understand the origin and convergent evolution of C4 and CAM plants and shed light on the adaptation of plants in dry, hot environments.

Dryopteriswulingshanensis (Dryopteridaceae), a new species from Hunan, China.

Dryopteriswulingshanensis, a new species growing on limestone in the Wulingshan Mountains, Hunan, China, is described and illustrated. This species is most similar to D.jishouensis and D.gymnophylla on general morphological traits, such as the form of scales, rhizome and sori, but differs by the number of vascular bundles at the base of the petiole, length to width ratio of lamina, stalk length of basal pinnae, division of the lamina, apex form of the pinnule and habitat. Moreover, molecular phylogenetic analysis using the chloroplast rbcL gene suggested that D.wulingshanensis, as the sister group of D.jishouensis, is a monophyletic clade. According to its restricted geographic range, small populations and few individuals, D.wulingshanensis should be considered endangered, according to the IUCN Red List criteria.

Morphology and molecules support the new monotypic genus Fenghwaia (Rhamnaceae) from south China.

Fenghwaia, a new monotypic genus, along with the new species Fenghwaia gardeniicarpa, is described from Guangdong Province, China. The combined features of inferior ovary, cylindrical drupaceous fruits and orbicular and dorsiventrally-compressed seeds with an elongate and pronounced basal appendage make the new genus significantly different from other genera of the family. In addition, its pollen morphology also showed great similarity to other species of this stenopalynous family. The molecular phylogenetic analysis, based on nuclear ribosomal internal transcribed spacer (ITS) and plastid trnL-F intron spacer (trnL-F) DNA sequence data from the new genus and the other 375 species representing 58 genera of Rhamnaceae, indicates that Fenghwaia is nested within the 'rhamnoid' group and sister to the tribe Rhamneae and then both sister to the tribe Maesopsideae. A taxonomic classification key to the 'rhamnoid' group is provided, based on morphological characters. A global conservation assessment is also performed and classifies Fenghwaia gardeniicarpa as Near Threatened (NT).

Phylogeny and Taxonomy on Cryptic Species of Forked Ferns of Asia.

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.

Transcriptomic Evidence of Adaptive Evolution of the Epiphytic Fern Asplenium nidus.

Epiphytic ferns have been found to flourish after angiosperms dominated forest communities, and they play important roles in rainforest canopies. How do epiphytic ferns adapt to tropical rainforest canopy habitats? At present, we know little about the molecular mechanism underlying this adaptation. Asplenium nidus is a well-known epiphytic fern that is closely related to the terrestrial species Asplenium komarovii. Here, RNA-seq and comparative transcriptomic analyses were performed to explore the underlying basis of the adaptation of A. nidus to extreme environments. A total of 44.04 and 44.57 Mb clean reads were obtained from A. nidus and A. komarovii, respectively, and they were assembled into 89,741 and 77,912 unigenes. Functional annotation showed that 52,305 (58.28% of the total genes for A. nidus) and 45,938 (58.96% of the total genes for A. komarovii) unigenes were annotated in public databases. Genes involved in stress responses and photosynthesis were found to have undergone positive selection in A. nidus. Compared to A. komarovii, transcription factors related to stress response, leaf development, and root development were found to be considerably expanded in A. nidus, especially in the ANR1 subclade of MADS-box family genes which played roles in lateral root development. This study improves our understanding of the adaptation of A. nidus to epiphytic habitats by forming unique strategies.

Large-scale phylogenomic analysis resolves a backbone phylogeny in ferns.

Background: 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. Results: 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. Conclusions: 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.

Re-establishment of species from synonymies based on DNA barcoding and phylogenetic analysis using Diplopterygium simulans (Gleicheniaceae) as an example.

Because synonymy treatment traditionally relies on morphological judgments, it usually causes many problems in species delimitation and in the biodiversity catalogue. For example, Diplopterygium simulans, which belongs to the Gleicheniaceae family, has been considered to be synonymous with D. glaucum or D. giganteum based mainly on the morphology of its pinna rachis and blade. In the absence of molecular evidence, these revisions remain doubtful. DNA barcoding, which is considered to be a powerful method for species-level identification, was employed to assess the genetic distance among 9 members of the Diplopterygium genus. The results indicate that D. simulans is an independent species rather than a synonymy of D. glaucum or D. giganteum. Moreover, phylogenetic analysis uncovered the sisterhood of D. simulans and D. cantonense, which is supported by their geographical distributions and morphological traits. Incorrect synonymy treatment is prevalent in the characterization of biological diversity, and our study proposes a convenient and effective method for validating synonym treatments and discovering cryptic species.