Angiopterisnodosipetiolata (Marattiaceae), a new fern species from Yunnan, China.

Angiopterisnodosipetiolata Ting Wang tris, H.F.Chen & Y.H.Yan, a new fern of Marattiaceae, is described and illustrated. Morphologically, A.nodosipetiolata is similar to A.chingii with more than one naked pulvinus on the stipe and numerous jointed hairs on the undersides of the mature pinnae. However, the pinnae of A.nodosipetiolata are lanceolate and can reach up to 4-6 pairs, whereas they are elliptic and occur in 2-3 pairs in A.chingii. Phylogenetic and genetic distance analysis, based on the plastid genomes, also indicates that A.nodosipetiolata is not closely related to A.chingii. Currently, there are ca. 500 mature individuals in Gulinqing Nature Reserve and we suggest A.nodosipetiolata should be categorised as an Endangered (EN) species according to the criteria of IUCN.

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.

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.

Simplified Genomic Data Revealing the Decline of Aleuritopteris grevilleoides Population Accompanied by the Uplift of Dry-Hot Valley in Yunnan, China.

Understanding the evolutionary history of endangered species is crucial for identifying the main reasons for species endangerment in the past and predicting the changing trends and evolutionary directions of their future distribution. In order to study the impact of environmental changes caused by deep valley incision after the uplift of the Qinghai-Tibet Plateau on endangered species, we collected 23 samples belonging to four populations of Aleuritopteris grevilleoides, an endangered fern endemic to the dry-hot valleys (DHV) of Yunnan. Single-nucleotide variation sites (SNPs) were obtained by the genotyping-by-sequencing (GBS) method, and approximately 8085 SNP loci were identified. Through the reconstruction and analysis of genetic diversity, population structure, population dynamics, evolution time, and ancestral geographical distribution, combined with geological historical events such as the formation of dry-hot valleys, this study explores the formation history, current situation, reasons for endangerment and scientifically sound measures for the protection of A. grevilleoides. In our study, A. grevilleoides had low genetic diversity (Obs_Het = 0.16, Exp_Het = 0.32, Pi = 0.33) and a high inbreeding coefficient (Fis = 0.45). The differentiation events were 0.18 Mya, 0.16 Mya, and 0.11 Mya in the A. grevilleoides and may have been related to the formation of terraces within the dry-hot valleys. The history of population dynamics results shows that the diversion of the river resulted in a small amount of gene flow between the two clades, accompanied by a rapid increase in the population at 0.8 Mya. After that, the effective population sizes of A. grevilleoides began to contract continuously due to topographic changes resulting from the continuous expansion of dry-hot valleys. In conclusion, we found that the environmental changes caused by geological events might be the main reason for the changing population size of A. grevilleoides.

Phylogeny, character evolution, and biogeography of the fern genus Bolbitis (Dryopteridaceae).

Bolbitis is a pantropical fern genus of Dryopteridaceae with ca. 80 species mainly in tropical Asia. Earlier studies confirmed the monophyly of Bolbitis when Mickelia is excluded and identified three major clades in Bolbitis. However, earlier studies are based on relatively small sampling and the majority of Asian species are not sampled. In this study, DNA sequences of three plastid markers of 169 accessions representing ca. 68 (85 % of total) species of Bolbitis in nine out of the 10 series recognized by Hennipman (1977), and 54 accessions representing the five remaining bolbitidoid genera are used to infer a global phylogeny with a focus on Asian species. The major results include: (1) Bolbitis is strongly supported as monophyletic; (2) species of Bolbitis are resolved into four major clades and their relationships are: the Malagasy/Mascarene clade is sister to the rest, followed by the African clade which is sister to the American clade + the Asian clade; (3) six well-supported subclades are identified in the most speciose Asian clade; (4) the free-veined Egenolfia is embedded in Bolbitis and is paraphyletic in relation to species with anastomosing venation; (5) three series sensu Hennipman (1977), B. ser. Alienae, B. ser. Egenolfianae, and B. ser. Heteroclitae, are paraphyletic or polyphyletic; (6) evolution of six morphological characters is analyzed and free venation is found to have evolved from anastomosing venation and reversed to free venation in Bolbitis; and (7) biogeographical implications are drawn and it is shown that a single recent dispersal from Asia resulted in continental disjunction of closely related ferns of Bolbitis between Africa and America.

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.

Population Diversity Analysis Provide Insights into Provenance Identification of Dendrobium catenatum.

Dendrobium catenatum (Dendrobium officinale) is a valuable genuine herb. The source of this species is difficult to be identified by traditional methods including morphology, spectroscopy, and chromatography. We used the restriction site-associated DNA sequencing (RAD-seq) approach to perform the high-throughput sequencing of 24 D. catenatum provenances. In this study, 371.18 Gb clean data were obtained, and 655,057 high-quality SNPs were selected after their filtration. We used phylogenetic tree, genetic structure, and principal component analyses to examine the genetic diversities and genetic relationships of the 109 accessions. We found that D. catenatum could be divided into two groups, and each group was closely related to the distribution of the sampling sites. At the population level, the average nucleotide diversity (π) of the D. catenatum population mutation parameters was 0.1584 and the expected heterozygosity (HE) was 0.1575. The GXLPTP07 accessions showed the highest genetic diversity in terms of the private allele number, observed heterozygosity, and nucleotide diversity. The Mantel test showed a significant positive correlation between the genetic and geographic distances among the overall distribution. A genetic information database of D. catenatum was established, which confirmed that RAD-seq technology has the potential to be applied in the identification of medicinal Dendrobium of different origins.

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.

Ceratopteris chunii and Ceratopteris chingii (Pteridaceae), two new diploid species from China, based on morphological, cytological, and molecular data.

Understanding how natural hybridization and polyploidizations originate in plants requires identifying potential diploid ancestors. However, cryptic plant species are widespread, particularly in Ceratopteris (Pteridaceae). Identifying Ceratopteris cryptic species with different polyploidy levels is a challenge because Ceratopteris spp. exhibit high degrees of phenotypic plasticity. Here, two new cryptic species of Ceratopteris, Ceratopteris chunii and Ceratopteris chingii, are described and illustrated. Phylogenetic analyses reveal that each of the new species form a well-supported clade. C. chunii and C. chingii are similar to Ceratopteris gaudichaudii var. vulgaris and C. pteridoides, respectively, but distinct from their relatives in the stipe, basal pinna of the sterile leaf or subelliptic shape of the fertile leaf, as well as the spore surface. In addition, chromosome studies indicate that C. chunii and C. chingii are both diploid. These findings will help us further understand the origin of Ceratopteris polyploids in Asia.

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.

Genome size evolution of the extant lycophytes and ferns.

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.

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.

Dipterisshenzhenensis, a new endangered species of Dipteridaceae from Shenzhen, southern China.

Dipterisshenzhenensis, a new species of ferns from Shenzhen, Guangdong, southern China, is identified and described. It closely resembles D.chinensis but possesses several unique traits, such as long rhizome scales, castaneous stipe, and abaxially pale fronds with two fan-shaped fronds connected by a broad wing. Molecular evidence showed that D.shenzhenensis is allied to D.conjugata, whereas it has morphologically significant differences (P < 0.05) on the basis of quantitative trait statistical analysis. Overall, the morphological evidence, taken together with the result of cpDNA indicated that D.shenzhenensis is a distinct species.

Finding missing diversity from synonyms of Haplopteris (Pteridaceae).

Although taxonomists target the remote wild regions to discover new species, taxa lacking a comprehensive and modern systematic treatment may be the new hotspot for biodiversity discovery. The development of molecular systematics integrated with microscopic observation techniques has greatly improved the ability of taxonomists to identify species correctly. Vittariacentrochinensis Ching ex J.F. Cheng, regarded as a synonym of Haplopterisfudzinoi (Makino) E.H.Crane, remained hidden from the eyes of fern taxonomists for more than 20 years. Herein, we collected several population samples of V.centrochinensis by performing molecular phylogenetic analysis of five cpDNA regions (rbcL, atpA, matK, ndhF, and trnL-trnF) and through micromophological observation of specimens which differs from H.fudzinoi by lamina width and exospores. Considering the differences in morphology, geographical range, and genetic distance between these two species, we formally recognized V.centrochinensis as an authentic species and proposed a new combination Haplopteriscentrochinensis (Ching ex J.F.Cheng) Y.H.Yan, Z.Y.Wei & X.C.Zhang, comb. nov. Our findings demonstrate that several taxa in synonyms are missing, and nowadays taxonomy should also include re-evaluation of the past taxonomy.

Rediscovery of Angiopteris tonkinensis (Marattiaceae) after 100 years, and its revision.

The border area between south-eastern Yunnan, China and northern Vietnam is one of the regions with richest biological diversity including that of the fern genus Angiopteris (Marattiaceae). Based on the analysis of morphology and DNA sequences of multiple chloroplast regions (atpB, rbcL, rps4-trnS spacer and trnL-F spacer), we revised Angiopteris tonkinensis (Hayata) J.M.Camus and proposed a new combination Angiopteris tamdaoensis (Hayata) J.Y.Xiang & T.Wang, comb. nov., which was previously regarded as a synonym of A. tonkinensis. We found support for a monophyletic Angiopteris including Protomarattia. This discovery adds two new distribution sites of A. tonkinensis, one in China (Malipo, Yunnan) and one in Vietnam (Quan Ba, Ha Giang). We suggest A. tonkinensis should be categorised as Critically Endangered (CR) species according to the criteria of IUCN.

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.

Dating Whole Genome Duplication in Ceratopteris thalictroides and Potential Adaptive Values of Retained Gene Duplicates.

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.

The identity of Hypolepis robusta, as a new synonym of Hypolepis alpina (Dennstaedtiaceae), based on morphology and DNA barcoding and the new distribution.

Based on field observations and examinations of herbarium specimens (including type material), consulting the original literature and molecular phylogenetic analysis of the rbcL and trnL-F sequences, it is concluded that Hypolepis robusta is conspecific with Hypolepis alpina and is here formally treated as a synonym of it. Additionally H. alpina is reported with new distribution records in Guangdong, Guangxi and the Hainan Island of China, respectively.