Taxonomic significance of megaspores in some species of Selaginella from Arunachal Pradesh in India.

The present study was carried out to record the color, size, and ornamentation of megaspores in 18 species of the family Selaginellaceae from Arunachal Pradesh using scanning electron microscope (SEM) and light microscope (LM). Electron microscopic study of the spore features revealed the type of ornamentation and microsculptural detail. SEM study on megaspore of Selaginella pentagona (S. pentagona), Selaginella tenuifolia, Selaginella semicordata, and Selaginella chrysorrhizos is presented for the first time. Variation in the megaspore ornamentation is noted at the interspecific level and intraspecific level in some cases. Examination of the megaspores under study found all the megaspores as trilete, with a size ranging from 116 to 560 µm in diameter. Taxonomic key is prepared to differentiate the species. Short descriptions of megaspores are provided and supported by photo plates. New features are recorded for seven species. The study contributes to the separation of species within the genus Selaginella based on the spore feature and brings forward the use of spore as a diagnostic tool in the taxonomy of the genus. These works contribute to the systematic of the family Selaginellaceae and provide useful information in the field of palynology. RESEARCH HIGHLIGHTS: Study on megaspore features of Selaginella species collected from Arunachal Pradesh, using SEM and LM. Taxonomic key is provided for each species based on megaspores features. New megaspore features are recorded for the seven species. The study brings forward the use of spore as a diagnostic tool in the taxonomy of the genus. These works provide useful information in the field of systematic and palynology.

Plastid Genomes of the Early Vascular Plant Genus Selaginella Have Unusual Direct Repeat Structures and Drastically Reduced Gene Numbers.

The early vascular plants in the genus Selaginella, which is the sole genus of the Selaginellaceae family, have an important place in evolutionary history, along with ferns, as such plants are valuable resources for deciphering plant evolution. In this study, we sequenced and assembled the plastid genome (plastome) sequences of two Selaginella tamariscina individuals, as well as Selaginella stauntoniana and Selaginella involvens. Unlike the inverted repeat (IR) structures typically found in plant plastomes, Selaginella species had direct repeat (DR) structures, which were confirmed by Oxford Nanopore long-read sequence assembly. Comparative analyses of 19 lycophytes, including two Huperzia and one Isoetes species, revealed unique phylogenetic relationships between Selaginella species and related lycophytes, reflected by structural rearrangements involving two rounds of large inversions that resulted in dynamic changes between IR and DR blocks in the plastome sequence. Furthermore, we present other uncommon characteristics, including a small genome size, drastic reductions in gene and intron numbers, a high GC content, and extensive RNA editing. Although the 16 Selaginella species examined may not fully represent the genus, our findings suggest that Selaginella plastomes have undergone unique evolutionary events yielding genomic features unparalleled in other lycophytes, ferns, or seed plants.

Plastome-based phylogenomics resolves the placement of the sanguinolenta group in the spikemoss of lycophyte (Selaginellaceae).

Selaginellaceae have been shown to be monophyletic in previous studies, and include only the single genus Selaginella. However, the two most recent classifications of the genus disagree in terms of the number of subgenera recognized, and the position of problematic clades such as the "sanguinolenta" group, which has been resolved in quite different positions in different studies. Here, we performed a plastid-genome based phylogenomic analysis of Selaginellaceae to address this problem. The sanguinolenta group, represented here by three species, was resolved as sister to the remaining members of subg. Stachygynandrum. Additionally, subg. Exaltatae, subg. Ericetorum, and subg. Gymnogynum in clade A clustered into a well supported monophyletic clade but with conflicting topology between subgenera inside, which is possibly attributed to the early divergence among them. We uncovered substantial variation in both synonymous (dS) and nonsynonymous (dN) substitution rate, and GC content in plastomes of Selaginellaceae. The values of dS, dN, and GC content were significantly higher than those of other lycophytes (Isoetaceae and Lycopodiaceae). We observed a significant positive correlation between the high GC content, and the elevated dS and dN rates. In addition, the dS and dN values inferred among branches of Selaginellaceae were extremely variable. Our data indicate that this unevenly distributed substitution rate likely reflected relaxed or intensified selection among different lineages, which is possibly related to the inconsistency of the subgeneric phylogenetic topologies of Selaginellaceae.

Virtual issue: Ecology and evolution of pteridophytes in the era of molecular genetics.

The past quarter-century has witnessed a revolution in our understanding of the phylogenetics, systematics, and ecology of pteridophytes (ferns and lycophytes), particularly due to the rapid accumulation of plastid sequence data and a renewed interest in the ecology of the sexual phase of the life cycle. We here compile 19 papers recently published in the Journal of Plant Research dealing with the biology of pteridophytes, grouped into six categories: (1) breeding systems, (2) species complexes and polyploidization, (3) fossil taxa, (4) gametophyte ecology, (5) systematics, (6) biodiversity. We hope this collection of papers will be of value to researchers interested in this fascinating group of plants.

An update and reassessment of fern and lycophyte diversity data in the Japanese Archipelago.

The fern and lycophyte flora of Japan comprising 721 native taxa (including subspecies and varieties) plus 371 interspecific hybrids was reassessed using a nearly comprehensively sampled distribution map at 10 km resolution vouchered by 216,687 specimens, up-to-date cytotaxonomic information covering 74% of the taxa, and an rbcL sequence dataset covering 97.9% of the taxa. Spatial distribution of species richness and phylogenetic diversity was visualized. Apomixis was observed in 11.0% of the native taxa whose reproductive modes are known. The number of sexually reproducing polyploid taxa (n = 199) is less than sexual diploids (n = 241), and 30 of them are evidently allopolyploid, in contrast with the low number of possible autopolyploids (n = 4). Apomictic taxa were found to have smaller latitudinal ranges than sexual taxa or taxa with multiple reproductive modes. A morphological character dataset in Lucid format is provided for taxonomic identification of the native taxa.

The adaptive value of heterospory: Evidence from Selaginella.

Heterospory was a pivotal evolutionary innovation for land plants, but it has never been clear why it evolved. We used the geographic distributions of 114 species of the heterosporous lycophyte Selaginella to explore the functional ecology of microspore and megaspore size, traits that would be correlated with many aspects of a species' regeneration niche. We characterized habitats at a global scale using leaf area index (LAI), a measure of foliage density and thus shading, and net primary productivity (NPP), a measure of growth potential. Microspore size tends to decrease as habitat LAI and NPP increase, a trend that could be related to desiccation resistance or to filtration of wind-borne particles by leaf surfaces. Megaspore size tends to increase among species that inhabit regions of high LAI, but there is an important interaction with NPP. This geographical pattern suggests that larger megaspores provide an establishment advantage in shaded habitats, although in open habitats, where light is less limiting, higher productivity of the environment seems to give an advantage to species with smaller megaspores. These results support previous theoretical arguments that heterospory was originally an adaptation to the increasing height and density of Devonian vegetative canopies that accompanied the diversification of vascular plants with leaves.

Cloning and Functional Characterization of Two 4-Coumarate: CoA Ligase Genes from Selaginella moellendorffii.

Selaginella is an extant lycopodiophyte genus, which is representative of an ancient lineage of tracheophytes. The important evolutionary status makes it a valuable resource for the study of metabolic evolution in vascular plants. 4-coumarate: CoA ligase (4CL) is the pivotal enzyme that controls the flow of carbon through the phenylpropanoid metabolic pathway into the specific lignin, flavonoid, and wall-bound phenolics biosynthesis pathways. Although 4CLs have been extensively characterized in other vascular plants, little is known of their functions in Selaginella. Here, we isolated two 4CL genes (Sm4CL1 and Sm4CL2) from Selaginella moellendorffii. Based on the enzymatic activities of the recombinant proteins, both of these genes encoded bona fide 4CLs. The 4CL isoforms in S. moellendorffii have different activities: Sm4CL2 was more active than Sm4CL1. The enzymatic properties and gene expression patterns indicated that the 4CL genes have been conserved in the evolution of vascular plants.

A subgeneric classification of Selaginella (Selaginellaceae).

PREMISE OF THE STUDY: The lycophyte family Selaginellaceae includes approximately 750 herbaceous species worldwide, with the main species richness in the tropics and subtropics. We recently presented a phylogenetic analysis of Selaginellaceae based on DNA sequence data and, with the phylogeny as a framework, the study discussed the character evolution of the group focusing on gross morphology. Here we translate these findings into a new classification. METHODS: To present a robust and useful classification, we identified well-supported monophyletic groups from our previous phylogenetic analysis of 223 species, which together represent the diversity of the family with respect to morphology, taxonomy, and geographical distribution. Care was taken to choose groups with supporting morphology. KEY RESULTS: In this classification, we recognize a single genus Selaginella and seven subgenera: Selaginella, Rupestrae, Lepidophyllae, Gymnogynum, Exaltatae, Ericetorum, and Stachygynandrum. The subgenera are all well supported based on analysis of DNA sequence data and morphology. A key to the subgenera is presented. CONCLUSIONS: Our new classification is based on a well-founded hypothesis of the evolutionary relationships of Selaginella, and each subgenus can be identified by a suite of morphological features, most of them possible to study in the field. Our intention is that the classification will be useful not only to experts in the field, but also to a broader audience.

Phylogeny of Selaginellaceae: There is value in morphology after all!

PREMISE OF THE STUDY: The cosmopolitan lycophyte family Selaginellaceae, dating back to the Late Devonian-Early Carboniferous, is notorious for its many species with a seemingly undifferentiated gross morphology. This morphological stasis has for a long time hampered our understanding of the evolutionary history of the single genus Selaginella. Here we present a large-scale phylogenetic analysis of Selaginella, and based on the resulting phylogeny, we discuss morphological evolution in the group. METHODS: We sampled about one-third of the approximately 750 recognized Selaginella species. Evolutionary relationships were inferred from both chloroplast (rbcL) and single-copy nuclear gene data (pgiC and SQD1) using a Bayesian inference approach. The morphology of the group was studied and important features mapped onto the phylogeny. KEY RESULTS: We present an overall well-supported phylogeny of Selaginella, and the phylogenetic positions of some previously problematic taxa (i.e., S. sinensis and allies) are now resolved with strong support. We show that even though the evolution of most morphological characters involves reversals and/or parallelisms, several characters are phylogenetically informative. Seven major clades are identified, which each can be uniquely diagnosed by a suite of morphological features. There is value in morphology after all! CONCLUSIONS: Our hypothesis of the evolutionary relationships of Selaginella is well founded based on DNA sequence data, as well as morphology, and is in line with previous findings. It will serve as a firm basis for further studies on Selaginella with respect to, e.g., the poorly known alpha taxonomy, as well as evolutionary questions such as historical biogeographic reconstructions.

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Molecular cloning and identification of divinyl ether synthases CYP74M1 and CYP74M3.

Nonclassical P450s of CYP74 family control the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. At least ten genes attributed to four novel CYP74 subfamilies have been revealed by the recent sequencing of the spikemoss Selaginella moellendorffii Hieron genome. Two of these genes CYP74M1 and CYP74M3 have been cloned in the present study. Both recombinant proteins CYP74M1 and CYP74M3 were active towards the 13(S)-hydroperoxides of α-linolenic and linoleic acids (13-HPOT and 13-HPOD, respectively) and exhibited the activity of divinyl ether synthase (DES). Products were analyzed by gas chromatography-mass spectrometry. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the (1)H NMR, 2D-COSY, HSQC and HMBC. CYP74M1 (SmDES1) specifically converted 13-HPOT to (11Z)-etherolenic acid and 13-HPOD to (11Z)-etheroleic acid. CYP74M3 (SmDES2) turned 13-HPOT and 13-HPOD mainly to etherolenic and etheroleic acids, respectively. CYP74M1 and CYP74M3 are the first DESs detected in non-flowering plants. The obtained results demonstrate the existence of the sophisticated oxylipin biosynthetic machinery in the oldest taxa of vascular plants.

Molecular phylogenetic study and expression analysis of ATP-binding cassette transporter gene family in Oryza sativa in response to salt stress.

ATP-binding cassette (ABC) transporter is a large gene superfamily that utilizes the energy released from ATP hydrolysis for transporting myriad of substrates across the biological membranes. Although many investigations have been done on the structural and functional analysis of the ABC transporters in Oryza sativa, much less is known about molecular phylogenetic and global expression pattern of the complete ABC family in rice. In this study, we have carried out a comprehensive phylogenetic analysis constructing neighbor-joining and maximum-likelihood trees based on various statistical methods of different ABC protein subfamily of five plant lineages including Chlamydomonas reinhardtii (green algae), Physcomitrella patens (moss), Selaginella moellendorffii (lycophyte), Arabidopsis thaliana (dicot) and O. sativa (monocot) to explore the origin and evolutionary patterns of these ABC genes. We have identified several conserved motifs in nucleotide binding domain (NBD) of ABC proteins among all plant lineages during evolution. Amongst the different ABC protein subfamilies, 'ABCE' has not yet been identified in lower plant genomes (algae, moss and lycophytes). The result indicated that gene duplication and diversification process acted upon these genes as a major operative force creating new groups and subgroups and functional divergence during evolution. We have demonstrated that rice ABCI subfamily consists of only half size transporters that represented highly dynamic members showing maximum sequence variations among the other rice ABC subfamilies. The evolutionary and the expression analysis contribute to a deep insight into the evolution and diversity of rice ABC proteins and their roles in response to salt stress that facilitate our further understanding on rice ABC transporters.

Evolutionary relationship and structural characterization of the EPF/EPFL gene family.

EPF1-EPF2 and EPFL9/Stomagen act antagonistically in regulating leaf stomatal density. The aim of this study was to elucidate the evolutionary functional divergence of EPF/EPFL family genes. Phylogenetic analyses showed that AtEPFL9/Stomagen-like genes are conserved only in vascular plants and are closely related to AtEPF1/EPF2-like genes. Modeling showed that EPF/EPFL peptides share a common 3D structure that is constituted of a scaffold and loop. Molecular dynamics simulation suggested that AtEPF1/EPF2-like peptides form an additional disulfide bond in their loop regions and show greater flexibility in these regions than AtEPFL9/Stomagen-like peptides. This study uncovered the evolutionary relationship and the conformational divergence of proteins encoded by the EPF/EPFL family genes.

Selaginella moellendorffii has a reduced and highly conserved expansin superfamily with genes more closely related to angiosperms than to bryophytes.

BACKGROUND: Expansins are plant cell wall loosening proteins encoded by a large superfamily of genes, consisting of four families named EXPA, EXPB, EXLA, and EXLB. The evolution of the expansin superfamily is well understood in angiosperms, thanks to synteny-based evolutionary studies of the gene superfamily in Arabidopsis, rice, and Populus. Analysis of the expansin superfamily in the moss Physcomitrella patens revealed a superfamily without EXLA or EXLB genes that has evolved considerably and independently of angiosperm expansins. The sequencing of the Selaginella moellendorffii genome has allowed us to extend these analyses into an early diverging vascular plant. RESULTS: The expansin superfamily in Selaginella moellendorffii has now been assembled from genomic scaffolds. A smaller (and less diverse) superfamily is revealed, consistent with studies of other gene families in Selaginella. Selaginella has an expansin superfamily, which, like Physcomitrella, lacks EXLA or EXLB genes, but does contain two EXPA genes that are related to a particular Arabidopsis-rice clade involved in root hair development. CONCLUSIONS: From sequence-based phylogenetic analysis, most Selaginella expansins lie outside the Arabidopsis-rice clades, leading us to estimate the minimum number of expansins present in the last common ancestor of Selaginella and angiosperms at 2 EXPA genes and 1 EXPB gene. These results confirm Selaginella as an important intermediary between bryophytes and angiosperms.

[RAPD analysis of 8 medicinal species of Selaginella].

OBJECTIVE: To identify 8 medicinal species of Selaginella with method of molecular marker and to explore their genetic relationship. METHODS: 8 primers were selected from 60 random primers to amplify the total DNA extracts of 17 samples with RAPD method and then the amplified were analyzed. RESULTS: A total of amplified 58 bands and RAPD map were obtained. Based on them, a Jaccard's genetic similarity matrix and a dendrogram for the 8 plants were established using SPSS 11.5 software. CONCLUSION: The method revealed distinct differences of different species and the variation of the same species of Selaginella from different habitats. It can provide with genetic proof for authentication of some species and units below species.

[Study on HPLC fingerprint characteristics of Selaginella plants].

OBJECTIVE: To establish a method to analyze HPLC fingerprint characteristics of 10 plants from Selaginella. METHOD: HPLC was applied for establishment of fingerprints, which were used to evaluate and distinguish the different species of Selaginella. RESULT: The different species from Selaginella showed different HPLC fingerprint characteristic. The samples of the same species but collected in different period, different environment or different locations showed certain difference in fingerprints CONCLUSION: 2 important mutual fingerprint peaks were found in the 10 plants of Selaginella species and 5 peaks can be used as "main fingerprint peaks". The dates of these peaks can used for assessment of phylogenetic relation among species and evaluation of quality.

Isolation and partial characterization of trehalose 6-phosphate synthase aggregates from Selaginella lepidophylla plants.

Trehalose 6-phosphate synthase was purified from Selaginella lepidophylla plants and three aggregates of the enzyme were found by molecular exclusion chromatography, ion exchange chromatography and electrophoresis. Molecular exclusion chromatography showed four activity peaks with molecular weights of 624, 434, 224 and 115 kDa. Ion exchange chromatography allowed three fractions to be separated with TPS activity which eluted at 0.35, 0.7 and 1 M KCl. Native PAGE of each pool had three protein bands with apparent M(r) 660, 440 and 200 kDa. Western blot results showed that anti-TPS antibody interacted with 115 and 67 kDa polypeptides; these polypeptides share peptide sequences as indicated by internal sequence data. The effects of pH and temperature on enzyme stability and activity were studied. For fractions eluted at 0.35 and 1.0 M KCl, the optimum pH is 5.5, while an optimum pH of 7.5 for 0.7 M fraction was found. The three fractions eluted from ion exchange chromatography were stable in a pH 5-11 range. Optimal temperatures were 25, 45 and 55 degrees C for 0.7, 0.35 and 1.0 M fractions, respectively. The 0.7 M KCl fraction showed highest stability in a temperature range of 25-60 degrees C, whereas the 0.35 M KCl fraction had the lowest in the same temperature range.

[Determination of amentoflavone in 11 species of Selaginella medicinal material by HPLC].

OBJECTIVE: To determine the content of amentoflavone in 11 species of Selaginella medicinal materials. METHODS: HPLC was used with kromasil (250 x 4. 6 mm, 5 microm) column. The mobile phase consisted of methanol and phosphate buffer. The gradient condition was 0-->28 min with methanol 50%-->58%, and 30-->35 min with methanol 58%-->70%. The column temperature was 40 degrees C. The flow rate was 1. 0 ml/min and the detection wavelength at 330 nm. RESULTS: Amentoflavone showed a good linearity in the range of 153 - 767 ng. The average recovery was 97.03% and RSD was 0. 80% (n = 5). CONCLUSION: The method was rapid, accurate and can be used for quality control of Selaginella medicinal materials.

[Investigations and studies on medicinal plants from Selaginellaceae in Hubei Province].

OBJECTIVE: To investigate the species, the distribution and the utilization of the medicinal plants from Selaginellaceae in Hubei Province. METHOD: Through field investigations and comparing the collected specimens and literatures, the classification and identification of the species in Hubei Province were studied. RESULT: The results indicated that 15 species of plants from Selaginellaceae in Hubei, including 14 medicinal and 1 newly recorded species existed. The distribution and use in folk medicine were investigated. And the morphological description of several species was appended. CONCLUSION: The results provided a basis for the exploitation and utilization of the medicinal plant resources of Selagingellaceae.

Variation in chromosome numbers, CMA bands and 45S rDNA sites in species of Selaginella (Pteridophyta).

BACKGROUND AND AIMS: Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus is known only by the occurrence of a dysploid series of n=7-12, and a low frequency of polyploids. Aiming to contribute to a better understanding of the structural chromosomal variability of this genus, different staining methods were applied in species with different chromosome numbers. METHODS: The chromosome complements of seven species of Selaginella were analysed and, in four of them, the distribution of 45S rDNA sites was determined by fluorescent in situ hybridization. Additionally, CMA/DA/DAPI and silver nitrate staining were performed to investigate the correlation between the 45S rDNA sites, the heterochromatic bands and the number of active rDNA sites. KEY RESULTS: The chromosome numbers observed were 2n=18, 20 and 24. The species with 2n=20 exhibited chromosome complement sizes smaller and less variable than those with 2n=18. The only species with 2n=24, S. convoluta, had relatively large and asymmetrical chromosomes. The interphase nuclei in all species were of the chromocentric type. CMA/DA/DAPI staining showed only a weak chromosomal differentiation of heterochromatic bands. In S. willdenowii and S. convoluta eight and six CMA+ bands were observed, respectively, but no DAPI+ bands. The CMA+ bands corresponded in number, size and location to the rDNA sites. In general, the number of rDNA sites correlated with the maximum number of nucleoli per nucleus. Ten rDNA sites were found in S. plana (2n=20), eight in S. willdenowii (2n=18), six in S. convoluta (2n=24) and two in S. producta (2n=20). CONCLUSIONS: The remarkable variation in chromosome size and number and rDNA sites shows that dramatic karyological changes have occurred during the evolution of the genus at the diploid level. These data further suggest that the two putative basic numbers of the genus, x=9 and x=10, may have arisen two or more times independently.

[Resources of medicinal pteridophyta in Fengyang Mountain and Baishanzu Nature Reservation].

Natural environment of Fengyang Mountain and Baishanzu Nature Reservation is superiority. The vegetation is preserved well and the plant resources is rich. There are 79 species medicinal pteridophyta in this area. According to potency, it can be divided into 16 types, such as medicines for relieving exterior syndrome, medicines for clearing away heat, antirheumatic, medicines for inducing diuresis, medicines for expelling the parasites, hemostasis medicines, medicines for activating blood circulation to remove blood stasis, medicines for restoring vital energy, medicines for calming the liver to stop the wind, etc.