Infraspecific Variation of Huperzine A and B in Icelandic Huperzia selago Complex.

The alkaloids huperzine A and huperzine B were originally isolated from the Chinese club moss Huperzia serrata. They are known inhibitors of acetylcholinesterase, and especially huperzine A shows pharmaceutical potential for the treatment of Alzheimer's disease. Its supply heavily relies on natural plant sources belonging to the genus Huperzia, which shows considerable interspecific huperzine A variations. Furthermore, taxonomic controversy remains in this genus, particularly in the Huperzia selago group. With focus on Icelandic H. selago taxa, we aimed to explore the relatedness of Huperzia species using multi-locus phylogenetic analysis, and to investigate correlations between huperzine A contents, morphotypes, and genotypes. Phylogenetic analysis was performed with five chloroplastic loci (the intergenic spacer between the photosystem II protein D1 gene and the tRNA-His gene, maturase K, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, tRNA-Leu, and the intergenic spacer region between tRNA-Leu and tRNA-Phe). Huperzine A and huperzine B contents were determined using an HPLC-UV method. The phylogenetic analysis suggests that previously proposed Huperzia appressa and Huperzia arctica should not be considered species, but rather subspecies of H. selago. Three genotypes of Icelandic H. selago were identified and presented in a haplotype networking diagram. A significantly (p < 0.05) higher amount of huperzine A was found in H. selago genotype 3 (264 - 679 µg/g) than genotype 1 (20 - 180 µg/g), where the former shows a typical green and reflexed "selago" morphotype. The huperzine A content in genotype 3 is comparable to Chinese H. serrata and a good alternative huperzine A source. Genotype 2 contains multiple morphotypes with a broad huperzine A content (113 - 599 µg/g). The content of huperzine B in Icelandic taxa (6 - 13 µg/g) is much lower than that in Chinese H. serrata (79 - 207 µg/g).

Global transcriptome analysis of Huperzia serrata and identification of critical genes involved in the biosynthesis of huperzine A.

BACKGROUND: Huperzia serrata (H. serrata) is an economically important traditional Chinese herb with the notably medicinal value. As a representative member of the Lycopodiaceae family, the H. serrata produces various types of effectively bioactive lycopodium alkaloids, especially the huperzine A (HupA) which is a promising drug for Alzheimer's disease. Despite their medicinal importance, the public genomic and transcriptomic resources are very limited and the biosynthesis of HupA is largely unknown. Previous studies on comparison of 454-ESTs from H. serrata and Phlegmariurus carinatus predicted putative genes involved in lycopodium alkaloid biosynthesis, such as lysine decarboxylase like (LDC-like) protein and some CYP450s. However, these gene annotations were not carried out with further biochemical characterizations. To understand the biosynthesis of HupA and its regulation in H. serrata, a global transcriptome analysis on H. Serrata tissues was performed. RESULTS: In this study, we used the Illumina Highseq4000 platform to generate a substantial RNA sequencing dataset of H. serrata. A total of 40.1 Gb clean data was generated from four different tissues: root, stem, leaf, and sporangia and assembled into 181,141 unigenes. The total length, average length, N50 and GC content of unigenes were 219,520,611 bp, 1,211 bp, 2,488 bp and 42.51%, respectively. Among them, 105,516 unigenes (58.25%) were annotated by seven public databases (NR, NT, Swiss-Prot, KEGG, COG, Interpro, GO), and 54 GO terms and 3,391 transcription factors (TFs) were functionally classified, respectively. KEGG pathway analysis revealed that 72,230 unigenes were classified into 21 functional pathways. Three types of candidate enzymes, LDC, CAO and PKS, responsible for the biosynthesis of precursors of HupA were all identified in the transcripts. Four hundred and fifty-seven CYP450 genes in H. serrata were also analyzed and compared with tissue-specific gene expression. Moreover, two key classes of CYP450 genes BBE and SLS, with 23 members in total, for modification of the lycopodium alkaloid scaffold in the late two stages of biosynthesis of HupA were further evaluated. CONCLUSION: This study is the first report of global transcriptome analysis on all tissues of H. serrata, and critical genes involved in the biosynthesis of precursors and scaffold modifications of HupA were discovered and predicted. The transcriptome data from this work not only could provide an important resource for further investigating on metabolic pathways in H. serrata, but also shed light on synthetic biology study of HupA.

Molecular phylogenetics and the morphology of the Lycopodiaceae subfamily Huperzioideae supports three genera: Huperzia, Phlegmariurus and Phylloglossum.

The generic classification of huperzioid Lycopodiaceae was tested using Bayesian inference and Maximum likelihood phylogenetic analyses of DNA sequences from four chloroplast loci for 119 taxa and optimisation of 29 morphological characteristics onto the phylogeny. Consistent with previous studies, the subfamilies Lycopodioideae and Huperzioideae are monophyletic and diagnosable by synapomorphies that correlate with differences in their life-histories. Within the Huperzioideae, the monophyly of the widely adopted genus Huperzia (excl. Phylloglossum) is poorly supported. Three clades of huperzioid Lycopodiaceae were recovered in all analyses of molecular data: Phylloglossum drummondii, Huperzia sensu stricto and Phlegmariurus sensu lato. These clades are strongly supported by morphological characters, including differences in spores, gametophytes, sporophyte macro-morphology, as well as growth habit and life-histories. Our findings indicate that either a one-genus (Huperzia s.l.) or a three-genus (Phylloglossum, Huperzia s.s. and Phlegmariurus s.l.) classification of huperzioid Lycopods are equally supported by molecular evidence, but a two-genus system (Huperzia s.l.+Phylloglossum) is not. We recommend recognising three genera in the huperzioid Lycopodiaceae, as this classification best reflects evolutionary, ecological, and morphological divergence within the lineage.

[The utility of trnH-psbA gene for phylogenetic analysis of Huperziaceae and plant barcoding].

OBJECTIVE: The purpose of study was to discover the phylogenetic relations and plant barcoding of 17 plants from Huperziaceae. METHODS: Phylogenetic tree of chloroplast trnH-psbA gene of 17 plants from Huperziaceae was constructed by software. RESULTS: It showed that Huperziaceae could be divided into two genera Huperzia and Phlegmariurus and bootstrap value reached 91%. CONCLUSIONS: Holub and Qing' taxonomy was supported and 17 species in Huperziaceae were monophyletic groups and it suggested that trnH-psbA could be used as a DNA barcode to identify plants.

[Phylogeny relationship and molecular identification of ten Huperzia species (Huperziaceae) based on matK gene sequences].

OBJECTIVE: To study the phylogeny relationship and molecular identification of 10 species from Huperzia (Huperziaceae) based on matK gene sequences data. METHOD: Total DNA of nine species from Huperzia was extracted; matK gene sequence was amplified by PCR. PCR product was directly sequenced after purification. RESULT: The chloroplast matK gene nucleotide sequences from 9 species of Huperzia species were sequenced. The matK gene nucleotide sequences length was 1 589 bp. Analysis with Huperzia lucidula matK gene nucleotide sequences (download from GenBank) and taking Lycopodiella cernua as outgroup, Maximum Parsimony, Neighbor-Joining analyses and genetic distances were conducted using MEGA 3.1 software. 35 variable sites and 35 parsimony informative sites have been found. Pairwise genetic distances among 10 species of Huperzia was 1.59% - 0.25%. CONCLUSION: The results were consistent with the taxonomy in morphological of Huperzia. But H. longipetiolata and H. serrata were resolved into in different clade. There are 19 different sites of matK gene sequences between H. longipetiolata and H. serrata, the genetic distances is 0.121%. It is suggested H. longipetiolata should be as an independent species.

Molecular identification of twelve medicinal plants of Huperziaceae.

Twelve species of Huperziaceae have been identified for resolving the resource depletion of Huperziaceae plants. The chloroplast genes rbcL, rpL16, and psbA-trnH intergenic spacers were used in this study. Identification of different species of Huperziaceae is possible by analyzing the information obtained from rbcL, rpL16, and psbA-trnH intergenic spacer sequences. The result also can provide information with regard to the use of plant tissue culture and cuttage propagation to solve the problem of the resource depletion of these plants.

A survey of potential huperzine A natural resources in China: the Huperziaceae.

The Huperziaceae is comprised of two genera, Huperzia and Phlegmariurus. Because of the content of Lycopodium alkaloids like huperzine A, which are used to treat a number of human ailments, plants of the Huperziaceae are experiencing a rapid decline in China, mostly due to over-harvesting. Because of this trend, we engaged from 1995 to 2001 in an investigation of the natural resources of the Huperziaceae in China. The main objectives of this study were: to catalog Huperziaceae plant resources including the occurrence, general distribution, and abundance of the various Huperziaceae species in China; and to determine traditional use and pharmaceutical values of each species. Twenty-nine species, 2 varieties, and 2 forma of Huperzia and 19 species of Phlegmariurus were identified through field investigation, collection, visits with local traditional doctors, and review of specimens in herbaria and of the literature. Ethnobotanical studies of these plants revealed that 33 of these species are used by the local communities for medicinal purposes. One species, Huperzia serrata, is one of the most popular. As a result, it is observing the greatest decline, mandating a change in collection practice and general attitude towards these plants. Introduction of conservation plans and training of the local communities regarding appropriate collection practices of these plants and their marketing in China are required to reverse the trend of decline among these species. In addition, development of cultivation or other propagation practices, such as in vitro propagation, would have the added benefits of socio-economic uplift of the local communities and sustainability of this important source of huperzine A.

[FTIR spectra-principal component analysis of phenetic relationships of Huperzia serrata and its closely related species].

Huperzia serrata is an important medicinal plant. This species is rich in inner-specific variation with various closely related species, and their individuals are also small with few identification characters. In the present paper, the method of Fourier transform infrared spectrometer with an OMNI collector was applied to obtaining the infrared spectra of 16 leaf samples including Huperzia serrata and its five closely related species (Huperzia sutchueniana, Phlegmariurus mingchegensis, Lycopodium japonicum, Selaginella doederleinii, Selaginella heterostachys). Based on the indices of wave number-absorbance, the differences of the 16 infrared spectra were compared by the method of Principal Component Analysis (PCA). The results showed that there is good correspondence between the position relationship of PCA three-dimensional plot of the samples based on the indices of wave number-absorbance of FTIR spectra and their phenetic relationship. Therefore, the infrared spectra could be applied to identifying the samples of Huperzia serrata and its closely related species.