Signatures of Adaptation and Purifying Selection in Highland Populations of Dasiphora fruticosa.

High mountains harbor a considerable proportion of biodiversity, but we know little about how diverse plants adapt to the harsh environment. Here we finished a high-quality genome assembly for Dasiphora fruticosa, an ecologically important plant distributed in the Qinghai-Tibetan Plateau and lowland of the Northern Hemisphere, and resequenced 592 natural individuals to address how this horticulture plant adapts to highland. Demographic analysis revealed D. fruticosa underwent a bottleneck after Naynayxungla Glaciation. Selective sweep analysis of two pairs of lowland and highland populations identified 63 shared genes related to cell wall organization or biogenesis, cellular component organization, and dwarfism, suggesting parallel adaptation to highland habitats. Most importantly, we found that stronger purging of estimated genetic load due to inbreeding in highland populations apparently contributed to their adaptation to the highest mountain. Our results revealed how plants could tolerate the extreme plateau, which could provide potential insights for species conservation and crop breeding.

Diploid species phylogeny and evolutionary reticulation indicate early radiation of Ephedra in the Tethys coast.

Reconstructing a robust species phylogeny and disentangling the evolutionary and biogeographic history of the gymnosperm genus Ephedra, which has a large genome and rich polyploids, remain a big challenge. Here we reconstructed a transcriptome-based phylogeny of 19 diploid Ephedra species, and explored evolutionary reticulations in this genus represented by 50 diploid and polyploid species, using four low-copy nuclear and nine plastid genes. The diploid species phylogeny indicates that the Mediterranean species diverged first, and the remaining species split into three clades, including the American species (Clade A), E. rhytidosperma, and all other Asian species (Clade B). The single-gene trees placed E. rhytidosperma sister to Clade A, Clade B, or Clades A + B in similar proportions, suggesting that radiation and gene flow likely occurred in the early evolution of Ephedra. In addition, reticulate evolution occurred not only among the deep nodes, but also in the recently evolved South American species, which further caused difficulty in phylogenetic reconstruction. Moreover, we found that allopolyploid speciation was pervasive in Ephedra. Our study also suggests that Ephedra very likely originated in the Tethys coast during the late Cretaceous, and the South American Ephedra species have a single origin by dispersal from Mexico or North America.

Resolving interspecific relationships within evolutionarily young lineages using RNA-seq data: An example from Pedicularis section Cyathophora (Orobanchaceae).

Phylogenomics has shown great potential in resolving evolutionary relationships at different taxonomical levels. However, it remains controversial whether all orthologous genes under different selective pressures can be concatenated for phylogenomic reconstruction. Here we used sect. Cyathophora of Pedicularis, one of the most species-rich genera of angiosperms in the alpine and arctic regions of the Northern Hemisphere, as a model to investigate the efficiency of RNA-seq in resolving relationships of closely related congeneric species. Flower transcriptomes were sequenced for all species of sect. Cyathophora and two outgroup species. Forty-one highly conserved single-/low-copy nuclear genes and 1553 orthologous groups (OGs) were identified and concatenated into five datasets based on gene copy numbers and Ka/Ks values to reconstruct the phylogeny of section Cyathophora. We also tested how many genes minimally can resolve the interspecific relationships, and reconstructed the evolutionary history of some floral characters in sect. Cyathophora. The results showed that the five different datasets consistently resolved the interspecific relationships of sect. Cyathophora, and the interspecific relationships can be robustly reconstructed with maximal support when ⩾20 single-/low-copy nuclear genes or 25 OGs are used. Our study suggests that all OGs under different selective pressures can be concatenated for phylogenomic reconstruction, and provides a successful and efficient use of RNA-seq in reconstructing interspecific relationships of a non-model plant group with recent radiations.

Floral nectary, nectar production dynamics, and floral reproductive isolation among closely related species of Pedicularis.

Floral nectar is thought to be one of the most important rewards that attract pollinators in Pedicularis; however, few studies have examined variation of nectary structure and/or nectar secretion in the genus, particularly among closely related species. Here we investigated nectary morphology, nectar quality, and nectar production dynamics in flowers of Pedicularis section Cyathophora. We found a conical floral nectary at the base of the ovary in species of the rex-thamnophila clade. Stomata were found on the surface of the nectary, and copious starch grains were detected in the nectary tissues. In contrast, a semi-annular nectary was found in flowers of the species of the superba clade. Only a few starch grains were observed in tissues of the semi-annular nectary, and the nectar sugar concentration in these flowers was much lower than that in the flowers of the rex-thamnophila clade. Our results indicate that the floral nectary has experienced considerable morphological, structural, and functional differentiation among closely related species of Pedicularis. This could have affected nectar production, leading to a shift of the pollination mode. Our results also imply that variation of the nectary morphology and nectar production may have played an important role in the speciation of sect. Cyathophora.

Gapless genome assembly of azalea and multi-omics investigation into divergence between two species with distinct flower color.

The genus Rhododendron (Ericaceae), with more than 1000 species highly diverse in flower color, is providing distinct ornamental values and a model system for flower color studies. Here, we investigated the divergence between two parental species with different flower color widely used for azalea breeding. Gapless genome assembly was generated for the yellow-flowered azalea, Rhododendron molle. Comparative genomics found recent proliferation of long terminal repeat retrotransposons (LTR-RTs), especially Gypsy, has resulted in a 125 Mb (19%) genome size increase in species-specific regions, and a significant amount of dispersed gene duplicates (13 402) and pseudogenes (17 437). Metabolomic assessment revealed that yellow flower coloration is attributed to the dynamic changes of carotenoids/flavonols biosynthesis and chlorophyll degradation. Time-ordered gene co-expression networks (TO-GCNs) and the comparison confirmed the metabolome and uncovered the specific gene regulatory changes underpinning the distinct flower pigmentation. B3 and ERF TFs were found dominating the gene regulation of carotenoids/flavonols characterized pigmentation in R. molle, while WRKY, ERF, WD40, C2H2, and NAC TFs collectively regulated the anthocyanins characterized pigmentation in the red-flowered R simsii. This study employed a multi-omics strategy in disentangling the complex divergence between two important azaleas and provided references for further functional genetics and molecular breeding.

Phylogenetic relationships and generic delimitation of Eurasian Aster (Asteraceae: Astereae) inferred from ITS, ETS and trnL-F sequence data.

BACKGROUND AND AIMS: The classification and phylogeny of Eurasian (EA) Aster (Asterinae, Astereae, Asteraceae) remain poorly resolved. Some taxonomists adopt a broad definition of EA Aster, whereas others favour a narrow generic concept. The present study aims to delimit EA Aster sensu stricto (s.s.), elucidate the phylogenetic relationships of EA Aster s.s. and segregate genera. METHODS: The internal and external transcribed spacers of nuclear ribosomal DNA and the plastid DNA trnL-F region were used to reconstruct the phylogeny of EA Aster through maximum parsimony and Bayesian analyses. KEY RESULTS: The analyses strongly support an Aster clade including the genera Sheareria, Rhynchospermum, Kalimeris (excluding Kalimeris longipetiolata), Heteropappus, Miyamayomena, Turczaninowia, Rhinactinidia, eastern Asian Doellingeria, Asterothamnus and Arctogeron. Many well-recognized species of Chinese Aster s.s. lie outside of the Aster clade. CONCLUSIONS: The results reveal that EA Aster s.s. is both paraphyletic and polyphyletic. Sheareria, Rhynchospermum, Kalimeris (excluding K. longipetiolata), Heteropappus, Miyamayomena, Turczaninowia, Rhinactinidia, eastern Asian Doellingeria, Asterothamnus and Arctogeron should be included in Aster, whereas many species of Chinese Aster s.s. should be excluded. The recircumscribed Aster should be divided into two subgenera and nine sections. Kalimeris longipetiolata, Aster batangensis, A. ser. Albescentes, A. series Hersileoides, a two-species group composed of A. senecioides and A. fuscescens, and a six-species group including A. asteroides, should be elevated to generic level. With the Aster clade, they belong to the Australasian lineages. The generic status of Callistephus should be maintained. Whether Galatella (including Crinitina) and Tripolium should remain as genera or be merged into a single genus remains to be determined. In addition, the taxonomic status of A. auriculatus and the A. pycnophyllus-A. panduratus clade remains unresolved, and the systematic position of some segregates of EA Aster requires further study.

Potential allopolyploid origin of Ericales revealed with gene-tree reconciliation.

Few incidents of ancient allopolyploidization (polyploidization by hybridization or merging diverged genomes) were previously revealed, although there is significant evidence for the accumulation of whole genome duplications (WGD) in plants. Here, we focused on Ericales, one of the largest and most diverse angiosperm orders with significant ornamental and economic value. Through integrating 24 high-quality whole genome data selected from ~ 200 Superasterids genomes/species and an algorithm of topology-based gene-tree reconciliation, we explored the evolutionary history of in Ericales with ancient complex. We unraveled the allopolyploid origin of Ericales and detected extensive lineage-specific gene loss following the polyploidization. Our study provided a new hypothesis regarding the origin of Ericales and revealed an instructive perspective of gene loss as a pervasive source of genetic variation and adaptive phenotypic diversity in Ericales.

Chromosome-level genome assembly of a parent species of widely cultivated azaleas.

Azaleas (Ericaceae) comprise one of the most diverse ornamental plants, renowned for their cultural and economic importance. We present a chromosome-scale genome assembly for Rhododendron simsii, the primary ancestor of azalea cultivars. Genome analyses unveil the remnants of an ancient whole-genome duplication preceding the radiation of most Ericaceae, likely contributing to the genomic architecture of flowering time. Small-scale gene duplications contribute to the expansion of gene families involved in azalea pigment biosynthesis. We reconstruct entire metabolic pathways for anthocyanins and carotenoids and their potential regulatory networks by detailed analysis of time-ordered gene co-expression networks. MYB, bHLH, and WD40 transcription factors may collectively regulate anthocyanin accumulation in R. simsii, particularly at the initial stages of flower coloration, and with WRKY transcription factors controlling progressive flower coloring at later stages. This work provides a cornerstone for understanding the underlying genetics governing flower timing and coloration and could accelerate selective breeding in azalea.

The study of transcriptome sequencing for flower coloration in different anthesis stages of alpine ornamental herb (Meconopsis 'Lingholm').

Meconopsis (Papaveraceae) is an interesting alpine herb, mainly distributed in the mountainous area of southwest China and high altitude zone in Tibetan-Himalaya. Different Meconopsis species showed a flower color alteration in different anthesis stages, Meconopsis 'Lingholm' is one of the localized species whose petal color changes from purple to blue during the flowering process. In general, the blue color flower is a rare kind, and usually hard to cultivate artificially. The molecular mechanism of flower color formation and color alteration of alpine flowers were reported by many research workers. To find critical genes that regulate Meconopsis 'Lingholm' color alteration and the mechanism of environmental adaptation, the current study performed transcriptome sequencing by using Meconopsis 'Lingholm' petals from different anthesis stages. There were totally 91,615 unigenes obtained from 31.4 Gb sequencing data, and differentially expressed genes between two consecutive flowering stages were obtained. Bioinformatics studies showed genes regulating petal color alteration were activated. Moreover, the functional analysis showed that Meconopsis 'Lingholm' showed a stress response to mechanical damage, non-biological stimulation and water deficiency in the bud stage, as well as showed a stress response to the cold from cracking stage to blooming stage. Furthermore, RNA-Seq results were verified using nine randomly selected genes by qPCR, which showed same expression trend with sequencing results. During this study, 20 candidate genes identified for further studies, which included five petal color related genes and 15 environmental response genes.

Extensive population expansion of Pedicularis longiflora (Orobanchaceae) on the Qinghai-Tibetan Plateau and its correlation with the Quaternary climate change.

The Qinghai-Tibetan Plateau (QTP) is thought to be more strongly affected by the Quaternary glaciations than most other regions of the same latitude. It would be of great interest to investigate the population genetic structure of organisms distributed on the platform and its correlation with the Quaternary climatic oscillations. Here we used the chloroplast (cp)DNA trnT-trnF sequence to study genetic variation and phylogeography of Pedicularis longiflora, an alpine herb with extensive distribution on the QTP. Based on a range-wide sampling comprising 41 populations and 910 individuals, we detected 30 cpDNA haplotypes that were divided into five clades by phylogenetic and network analyses and a strong phylogeographical structure. All haplotypes but one in the three basal clades occur exclusively in the southeast QTP, whereas haplotypes in the young clade V occupy almost the whole species range. In particular, the young haplotype H18 occurs in 420 individuals, even at a frequency of 100% in some QTP platform populations and the Altai population. The haplotype distribution pattern, together with molecular clock estimation and mismatch distribution analysis, suggests that the southeast QTP was either a refuge for P. longiflora during the Quaternary climatic change or is the place of origin of the species. The present wide distribution of the species on the QTP platform has resulted from recent population expansions which could be dated back to 120,000-17,000 years ago, a period mostly before the last glacial maximum. The possible relationships among geographic genetic structure, climatic change and species diversification in Pedicularis are also discussed.

Identification of a novel missense mutation of MSX1 gene in Chinese family with autosomal-dominant oligodontia.

OBJECTIVES: Oligodontia is defined as the congenital absence of 6 or more permanent teeth excluding the third molar. The occurrence of non-syndromic still remains poorly understood, but in recent years some cases have been reported where mutations or polymorphisms of PAX9 and MSX1 had been associated with non-syndromic oligodontia. The objective of the present work was to study the phenotype and genotype of three generations of a Han Chinese family affected by non-syndromic autosomal-dominant oligodontia. DESIGN: We examined all individuals of the oligodontia family by clinical and radiographic examinations. Based on clinical manifestations, candidate genes MSX1 and PAX9 were picked up to analyse and screen mutations. RESULTS: Dental evaluation showed that the most commonly missing teeth are the mandibular second premolars, followed by the maxillary second premolars and maxillary lateral incisors, and subsequently the maxillary first premolars. The probability of missing a particular type of tooth is not always bilaterally symmetrical, and differences exist between maxilla and mandible. PCR-SSCP analysis and DNA sequencing revealed a novel missense mutation c.662C>A in a highly conserved homeobox sequence of MSX1 and a known polymorphisms c.347C>G. CONCLUSION: Our finding suggests the missense transversion (c.662C>A) and the polymorphisms (c.347C>G) may be responsible for oligodontia phenotype in this Chinese family.

Could calcitonin be a useful therapeutic agent for trigeminal neuralgia?

Trigeminal neuralgia (TN) has been recognized as one of the most common neurovascular syndromes caused by the vascular contact of the trigeminal nerve in its root entry zone (REZ) with a branch of the superior or anterior inferior cerebellar arteries, leading to a demyelinization of trigeminal sensory fibers within either the nerve root or, less commonly, the brainstem. There is a lack of certainty regarding the aetiology and pathophysiology of TN, therefore the treatment of trigeminal neuropathic pain disorders continues to be a major therapeutic challenge. The identification of novel therapeutic agents for the treatment of these disorders is important. Calcitonin (especially intranasal) provides an interesting analgesic effect in a series of painful conditions including reflex sympathetic dystrophy syndrome, adhesive capsulitis, ankylosing spondylitis, rheumatoid arthritis, vertebral crush fractures and metastasis, phantom limb pain, etc. Exogenous calcitonin is thought to cross the blood-brain barrier and to accumulate slowly in the brain, inducing analgesia once sufficient receptors are occupied. We hypothesize that calcitonin may has anti - trigeminal neuralgia properties. From the clinical point of use, the analgesic effect of calcitonin will be beneficial throughout the whole period of medical treatment of trigeminal neuralgia patients.

Calcitonin may be a useful therapeutic agent for osteoclastogenesis syndromes involving premature eruption of the tooth.

Tooth eruption is a complex and tightly regulated process that involves cells of the tooth organ and the surrounding alveolus. Recent researches have shown that tooth eruption depends on the presence of osteoclasts to create an eruption pathway through the alveolar bone. The most important physiologic role likely being at the eruptive site, in the formation of osteoclasts through signaling via the RANKL/OPG pathway. Calcitonin is an endogenous inhibitor of osteoclast development and function and thus of bone resorption. Specific calcitonin receptors are expressed on osteoclasts and their activation leads to the inhibition of osteoclast development and functions. Recent concepts about inhibiting osteoclastogenesis of calcitonin is that RANKL-induced osteoclastogenesis were blocked by the endogenous decoy receptor osteoprotegerin and were also strongly reduced by calcitonin, we hypothesize that calcitonin may has anti-eruption properties. For the clinical point of view, we can inject calcitonin in the oral mucosa of the affected tooth to inhibit bone resorption, then to facilitate root forming which may useful to premature eruption of tooth and short root anomaly disease (SRA) caused by every reasons such as hypoplasia of teeth root (HTR), Singleton-Mertern syndrome (SMS), infection and iatrogenic factors, etc.

Inhibition of RANK/RANKL signal transduction pathway: a promising approach for osteoporosis treatment.

Osteoporosis is a bone disease causing impaired bone strength. It is characterized by increased osteoclast formation or enhanced bone resorption, leading to an increased risk of fragility fractures. Its prevalence increases with age. The advent of an aging population suggests that progressively more individuals will develop this disease in the aging population. A number of drugs for the prevention and treatment of osteoporosis act by inhibiting bone resorption. However, the effectiveness of osteoporosis treatment in clinical practice is limited. Since the osteoclast is the only cell in the body that is capable of resorbing bone, understanding its biology will be necessary for developing a new therapeutic approach for osteoporosis. Recently, it was discovered that the receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system is an important signal transduction pathway that regulates osteoclast formation. The binding of OPG to RANKL inhibits the binding between RANKL and RANK; this, in turn, prevents osteoclast precursors from differentiating and fusing to form mature osteoclasts. Therefore, the inhibition of the RANK/RANKL pathway inhibits osteoclast formation, differentiation, activation, and bone resorption. A potential clinical antiresorptive therapy can be developed by using an anti-RANKL monoclonal antibody, such as denosumab, that binds to RANKL with high affinity and specificity and blocks RANKL-RANK interactions.

[Neural engineering and neural prostheses].

The motivation of the brain-computer interface (BCI) research and its potential applications are introduced in this paper. Some of the problems in BCI-based medical device developments are also discussed.

A multistage, multimethod approach for automatic detection and classification of epileptiform EEG.

An efficient system for detection of epileptic activity in ambulatory electroencephalogram (EEG) must be sensitive to abnormalities while keeping the false-detection rate to a low level. Such requirements could be fulfilled neither by single stage nor by simple method strategy, due to the extreme variety of EEG morphologies and frequency of artifacts. The present study proposes a robust system that combines multiple signal-processing methods in a multistage scheme, integrating adaptive filtering, wavelet transform, artificial neural network, and expert system. The system consists of two main stages: a preliminary screening stage in which data are reduced significantly, followed by an analytical stage. Unlike most systems that merely focus on sharp transients, our system also takes into account slow waves. A nonlinear filter for separation of nonstationary and stationary EEG components is also developed in this paper. The system was evaluated on testing data from 81 patients, totaling more than 800 hours of recordings. 90.0% of the epileptic events were correctly detected. The detection rate of sharp transients was 98.0% and overall false-detection rate was 6.1%. We conclude that our system has good performance in detecting epileptiform activities and the multistage multimethod approach is an appropriate way of solving this problem.

[Application of digital signal processing to the analysis of biomolecular sequences].

A preliminary introduction is given to the application of digital signal processing to the analysis of biomolecular sequences. How to transform a character string into a numerical sequence is discussed from the time domain and the frequency domain.

Molecular phylogeny of Asian Meconopsis based on nuclear ribosomal and chloroplast DNA sequence data.

The taxonomy and phylogeny of Asian Meconopsis (Himalayan blue poppy) remain largely unresolved. We used the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and the chloroplast DNA (cpDNA) trnL-F region for phylogenetic reconstruction of Meconopsis and its close relatives Papaver, Roemeria, and Stylomecon. We identified five main clades, which were well-supported in the gene trees reconstructed with the nrDNA ITS and cpDNA trnL-F sequences. We found that 41 species of Asian Meconopsis did not constitute a monophyletic clade, but formed two solid clades (I and V) separated in the phylogenetic tree by three clades (II, III and IV) of Papaver and its allies. Clade V includes only four Asian Meconopsis species, with the remaining 90 percent of Asian species included in clade I. In this core Asian Meconopsis clade, five subclades (Ia-Ie) were recognized in the nrDNA ITS tree. Three species (Meconopsis discigera, M. pinnatifolia, and M. torquata) of subgenus Discogyne were imbedded in subclade Ia, indicating that the present definition of subgenera in Meconopsis should be rejected. These subclades are inconsistent with any series or sections of the present classifications, suggesting that classifications of the genus should be completely revised. Finally, proposals for further revision of the genus Meconopsis were put forward based on molecular, morphological, and biogeographical evidences.

Phylogeographic evidence for a link of species divergence of Ephedra in the Qinghai-Tibetan Plateau and adjacent regions to the Miocene Asian aridification.

The Qinghai-Tibetan Plateau (QTP) has become one of the hotspots for phylogeographical studies due to its high species diversity. However, most previous studies have focused on the effects of the Quaternary glaciations on phylogeographical structures and the locations of glacial refugia, and little is known about the effects of the aridization of interior Asia on plant population structure and speciation. Here the chloroplast DNA (cpDNA) trnT-trnF and trnS-trnfM sequences were used to investigate the differentiation and phylogeographical history of 14 Ephedra species from the QTP and northern China, based on a sampling of 107 populations. The phylogeographical analysis, together with phylogenetic reconstruction based on combined four cpDNA fragments (rbcL, rpl16, rps4, and trnS-trnfM), supports three main lineages (eastern QTP, southern QTP, and northern China) of these Ephedra species. Divergence of each lineage could be dated to the Middle or Late Miocene, and was very likely linked to the uplift of the QTP and the Asian aridification, given the high drought and/or cold tolerance of Ephedra. Most of the Ephedra species had low intraspecific variation and lacked a strong phylogeographical structure, which could be partially attributed to clonal reproduction and a relatively recent origin. In addition, ten of the detected 25 cpDNA haplotypes are shared among species, suggesting that a wide sampling of species is helpful to investigate the origin of observed haplotypes and make reliable phylogeographical inference. Moreover, the systematic positions of some Ephedra species are discussed.

Great genetic differentiation among populations of Meconopsis integrifolia and its implication for plant speciation in the Qinghai-Tibetan Plateau.

The complex tectonic events and climatic oscillations in the Qinghai-Tibetan Plateau (QTP), the largest and highest plateau in the world, are thought to have had great effects on the evolutionary history of the native plants. Of great interest is to investigate plant population genetic divergence in the QTP and its correlation with the geologic and climatic changes. We conducted a range-wide phylogeographical analysis of M. integrifolia based on the chloroplast DNA (cpDNA) trnL-trnF and trnfM-trnS regions, and defined 26 haplotypes that were phylogenetically divided into six clades dated to the late Tertiary. The six clades correspond, respectively, to highly differentiated population groups that do not overlap in geographic distribution, implying that the mountain ranges acting as corridors or barriers greatly affected the evolutionary history of the QTP plants. The older clade of M. integrifolia only occurs in the southwest of the species' range, whereas the distributions of younger clades extend northeastward in the eastern QTP, suggesting that climatic divergence resulting from the uplift of the QTP triggered the initial divergence of M. integrifolia native to the plateau. Also, the nrDNA ITS region was used to clarify the unexpected phylogenetic relationships of cpDNA haplotypes between M. integrifolia and M. betonicifolia. The topological incongruence between the two phylogenies suggests an ancestral hybridization between the two species. Our study indicates that geographic isolation and hybridization are two important mechanisms responsible for the population differentiation and speciation of Meconopsis, a species-rich genus with complex polyploids.