Natural hybridization among three Rhododendron species (Ericaceae) revealed by morphological and genomic evidence.

BACKGROUND: Natural hybridization can influence the adaptive response to selection and accelerate species diversification. Understanding the composition and structure of hybrid zones may elucidate patterns of hybridization processes that are important to the formation and maintenance of species, especially for taxa that have experienced rapidly adaptive radiation. Here, we used morphological traits, ddRAD-seq and plastid DNA sequence data to investigate the structure of a Rhododendron hybrid zone and uncover the hybridization patterns among three sympatric and closely related species. RESULTS: Our results show that the hybrid zone is complex, where bi-directional hybridization takes place among the three sympatric parental species: R. spinuliferum, R. scabrifolium, and R. spiciferum. Hybrids between R. spinuliferum and R. spiciferum (R. ×duclouxii) comprise multiple hybrid classes and a high proportion of F1 generation hybrids, while a novel hybrid taxon between R. spinuliferum and R. scabrifolium dominated the F2 generation, but no backcross individuals were detected. The hybrid zone showed basically coincident patterns of population structure between genomic and morphological data. CONCLUSIONS: Natural hybridization exists among the three Rhododendron species in the hybrid zone, although patterns of hybrid formation vary between hybrid taxa, which may result in different evolutionary outcomes. This study represents a unique opportunity to dissect the ecological and evolutionary mechanisms associated with adaptive radiation of Rhododendron species in a biodiversity hotspot.

Incomplete reproductive isolation between Rhododendron taxa enables hybrid formation and persistence.

The evolutionary consequences of hybridization ultimately depend on the magnitude of reproductive isolation between hybrids and their parents. We evaluated the relative contributions of pre- and post-zygotic barriers to reproduction for hybrid formation, hybrid persistence and potential for reproductive isolation of hybrids formed between two Rhododendron species, R. spiciferum and R. spinuliferum. Our study established that incomplete reproductive isolation promotes hybrid formation and persistence and delays hybrid speciation. All pre-zygotic barriers to reproduction leading to hybrid formation are incomplete: parental species have overlapping flowering; they share the same pollinators; reciprocal assessments of pollen tube germination and growth do not differ among parents. The absence of post-zygotic barriers between parental taxa indicates that the persistence of hybrids is likely. Reproductive isolation was incomplete between hybrids and parents in all cases studied, although asymmetric differences in reproductive fitness were prevalent and possibly explain the genetic structure of natural hybrid swarms where hybridization is known to be bidirectional but asymmetric. Introgression, rather than speciation, is a probable evolutionary outcome of hybridization between the two Rhododendron taxa. Our study provides insights into understanding the evolutionary implications of natural hybridization in woody plants.

Characterisation of flower colouration in 30 Rhododendron species via anthocyanin and flavonol identification and quantitative traits.

Floral colour is a key reproductive character, often associated with environmental adaptation, and subject to human intervention. A large number of Rhododendron species differ widely in flower colour, providing a good model for flower colouration. The chromatic features and anthocyanin compositions of 30 species from seven subgenera were systematically analysed. The Royal Horticultural Society Colour Chart and CIE L*a*b* system were employed to describe and investigate flower colours. The UPLC-PDA/ESI-MSn system was used to identify and quantify anthocyanins in petal extracts. The flower colours of 30 Rhododendron species were categorised into four groups - red, purplish pink, purple and white. Seven anthocyanins were identified and quantified in petals: delphinidin, cyanidin and malvidin 3-O-arabinoside-5-O-glucosides, cyanidin 3,5-di-O-glucoside, 3-O-galactoside and 3-O-arabinoside, and delphinidin 3-O-glucoside. The red-flowered species mainly contained cyanidin monoglycosides and had much higher total anthocyanin content than purplish pink- and purple-flowered species. Purplish pink- and purple-flowered species had similar anthocyanin types and content. The chromatic differences were significant among groups, except the purplish pink and purple groups. Statistical analysis showed that Cy3Gal and Cy3Arb are characteristic for red-flowered species, and Mv3Arb5G and Dp3Arb5G play important roles in purple colouration; their contents were major components that greatly affected the chromatic parameters. In total, 21 flavonol derivates were identified. However, total flavonol content and co-pigmentation index showed no significant difference or correlation among/with colour groups, suggesting that flavonols might not play a major role in colouration. These results enhance our knowledge of the biochemical basis of flower colouration in Rhododendron species, and provide a foundation for genetic variation studies and aid in breeding cultivars with novel flower colours.

Rhododendron tomentosum (Ledum palustre). A review of traditional use based on current research.

Rhododendron tomentosum Harmaja (previously: Ledum palustre) is a fragrant evergreen shrub found in peaty soils in northern Europe, Asia and North America, commonly referred to as wild rosemary, marsh tea, marsh rosemary or northern Labrador tea. At least since the eighteenth century it has been used in ethnomedicine for the treatment of various ailments, such as rheumatism, cough, cold and insect bites, as well as a repellent. The essential oil of wild rosemary with the rich polyphenolic fraction possesses analgesic, anti-inflammatory, antimicrobial, antiviral, antifungal and insecticidal potential, demonstrated by in vivo and in vitro studies. In addition, recent scientific research reported the promising antidiabetic, antioxidant and anticancer properties. This review summarizes the information concerning taxonomy, botany, ecology, chemical composition, biological activities, toxicology and traditional and contemporary application of Rhododendron tomentosum plants.

Effects of nutrient addition on leaf chemistry, morphology, and photosynthetic capacity of three bog shrubs.

Plants in nutrient-poor environments typically have low foliar nitrogen (N) concentrations, long-lived tissues with leaf traits designed to use nutrients efficiently, and low rates of photosynthesis. We postulated that increasing N availability due to atmospheric deposition would increase photosynthetic capacity, foliar N, and specific leaf area (SLA) of bog shrubs. We measured photosynthesis, foliar chemistry and leaf morphology in three ericaceous shrubs (Vaccinium myrtilloides, Ledum groenlandicum and Chamaedaphne calyculata) in a long-term fertilization experiment at Mer Bleue bog, Ontario, Canada, with a background deposition of 0.8 g N m(-2) a(-1). While biomass and chlorophyll concentrations increased in the highest nutrient treatment for C. calyculata, we found no change in the rates of light-saturated photosynthesis (A(max)), carboxylation (V(cmax)), or SLA with nutrient (N with and without PK) addition, with the exception of a weak positive correlation between foliar N and A(max) for C. calyculata, and higher V(cmax) in L. groenlandicum with low nutrient addition. We found negative correlations between photosynthetic N use efficiency (PNUE) and foliar N, accompanied by a species-specific increase in one or more amino acids, which may be a sign of excess N availability and/or a mechanism to reduce ammonium (NH(4)) toxicity. We also observed a decrease in foliar soluble Ca and Mg concentrations, essential minerals for plant growth, but no change in polyamines, indicators of physiological stress under conditions of high N accumulation. These results suggest that plants adapted to low-nutrient environments do not shift their resource allocation to photosynthetic processes, even after reaching N sufficiency, but instead store the excess N in organic compounds for future use. In the long term, bog species may not be able to take advantage of elevated nutrients, resulting in them being replaced by species that are better adapted to a higher nutrient environment.

Hydraulic plasticity and limitations of alpine Rhododendron species.

In the European Alps, Rhododendron ferrugineum grows in silicate regions while Rhododendron hirsutum is restricted to limestone areas. At geologically mixed sites, also hybrids (Rhododendron × intermedium) can occur. We hypothesised that hydraulic properties would vary with the species' habitat requirements. Key hydraulic parameters (vulnerability to drought-induced embolism, stomata regulation) and related wood characteristics as well as diurnal courses of water potential (Ψ) and stomatal conductance were analysed on plants growing on a silicate, a limestone and a geologically mixed site. Highest embolism resistance[Ψ at 50% loss of conductivity (Ψ (50)), -3.24 ± 0.18 MPa] and the highest safety margin between the Ψ at stomata closure (Ψ (SC) at 10% of maximal leaf conductance) and Ψ (50) were observed in R. hirsutum at the limestone site (1.57 MPa). Like in R. ferrugineum, hydraulic parameters indicated less resistance at the geologically mixed site. Highest Ψ (50) (-1.95 ± 0.12 MPa), corresponding to wide conduits and a reduced conduit wall reinforcement, was found in R. × intermedium. Diurnal courses indicated a rapid stomata closure in response to low Ψ in R. hirsutum and R. × intermedium. The plasticity in drought adaptation of R. hirsutum corresponds to its ability to colonise dry limestone areas. In contrast, hydraulic limitations of R. × intermedium may explain restrictions to rather moist sites. This study provides insight into the role of xylem hydraulics and stomata regulation in shrub water relations, interspecific and site-specific differences in drought adaptation, as well as effects of hybridisation on plant hydraulics.

Asymmetric hybridization in Rhododendron agastum: a hybrid taxon comprising mainly F1s in Yunnan, China.

BACKGROUND AND AIMS: Rhododendron (Ericaceae) is a large woody genus in which hybridization is thought to play an important role in evolution and speciation, particularly in the Sino-Himalaya region where many interfertile species often occur sympatrically. Rhododendron agastum, a putative hybrid species, occurs in China, western Yunnan Province, in mixed populations with R. irroratum and R. delavayi. METHODS: Material of these taxa from two sites 400 km apart (ZhuJianYuan, ZJY and HuaDianBa, HDB) was examined using cpDNA and internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) loci, to test the possibility that R. agastum was in fact a hybrid between two of the other species. Chloroplast trnL-F and trnS-trnG sequences together distinguished R. irroratum, R. delavayi and some material of R. decorum, which is also considered a putative parent of R. agastum. KEY RESULTS: All 14 R. agastum plants from the HDB site had the delavayi cpDNA haplotype, whereas at the ZJY site 17 R. agastum plants had this haplotype and four had the R. irroratum haplotype. R. irroratum and R. delavayi are distinguished by five unequivocal point mutations in their ITS sequences; every R. agastum accession had an additive pattern (double peaks) at each of these sites. Data from AFLP loci were acquired for between ten and 21 plants of each taxon from each site, and were analysed using a Bayesian approach implemented by the program NewHybrids. The program confirmed the identity of all accessions of R. delavayi, and all R. irroratum except one, which was probably a backcross. All R. agastum from HDB and 19 of 21 from ZJY were classified as F1 hybrids; the other two could not be assigned a class. CONCLUSIONS: Rhododendron agastum represents populations of hybrids between R. irroratum and R. delavayi, which comprise mostly or only F1s, at the two sites examined. The sites differ in that at HDB there was no detected variation in cpDNA type or hybrid class, whereas at ZJY there was variation in both.

Dendrochronological potential of the alpine shrub Rhododendron nivale on the south-eastern Tibetan Plateau.

BACKGROUND AND AIMS: Shrubs and dwarf shrubs are wider spread on the Tibetan Plateau than trees and hence offer a unique opportunity to expand the present dendrochronological network into extreme environments beyond the survival limit of trees. Alpine shrublands on the Tibetan Plateau are characterized by rhododendron species. The dendrochronological potential of one alpine rhododendron species and its growth response to the extreme environment on the south-east Tibetan Plateau were investigated. METHODS: Twenty stem discs of the alpine snowy rhododendron (Rhododendron nivale) were collected close to the tongue of the Zuoqiupu Glacier in south-east Tibet, China. The skeleton plot technique was used for inter-comparison between samples to detect the growth pattern of each stem section. The ring-width chronology was developed by fitting a negative exponential function or a straight line of any slope. Bootstrapping correlations were calculated between the standard chronology and monthly climate data. KEY RESULTS: The wood of snowy rhododendron is diffuse-porous with evenly distributed small-diameter vessels. It has well-defined growth rings. Most stem sections can be visually and statistically cross-dated. The resulting 75-year-long standard ring-width chronology is highly correlated with a timberline fir chronology about 200 km apart, providing a high degree of confidence in the cross-dating. The climate/growth association of alpine snowy rhododendron and of this timberline fir is similar, reflecting an impact of monthly mean minimum temperatures in November of the previous year and in July during the year of ring formation. CONCLUSIONS: The alpine snowy rhododendron offers new research directions to investigate the environmental history of the Tibetan Plateau in those regions where up to now there was no chance of applying dendrochronology.

Authentication of the 31 species of toxic and potent Chinese Materia Medica by light microscopy, Part 3: two species of T/PCMM from flowers and their common adulterants.

Toxic and potent Chinese Materia Medica (T/PCMM) has become a hot and sensitive topic as more and more people around the world are interested in the safety of herbal medicines. T/PCMM is irreplaceable in treating some diseases; but it can easily cause serious problems if confused with other herbal medicines. Accurate identification is essential to ensure their safe use, but up to now, the literature on the authentication of T/PCMM is scant. Thus, we are undertaking a study of 31 T/PCMM originating from plants, animals, minerals, and secreta. Our previous study established microscopic observation as a simple, fast, accurate, and convenient method for identifying and authenticating animal and seed T/PCMM. This study focused on the authentication of flower T/PCMM as a part of the whole study. The flower T/PCMM studies were derived from two species, Datura metel L. (Flos Daturae) and Rhododendron molle G. Don (Flos Rhododendri Mollis). Other species easily confused with these two were also examined and characterized. Using the microscope camera, normal light and polarized light microscopy, we determined the macroscopic and microscopic features of the flowers; in addition, the oil immersion lens was used to study the pollen grain characteristics. The results demonstrated that flower T/PCMM can be identified and authenticated using a light microscope equipped with an oil immersion lens. This same equipment can be easily used to characterize other herbal flower medicines.

Hybridization and asymmetric introgression between Rhododendron eriocarpum and R. indicum on Yakushima Island, southwest Japan.

We examined natural hybridization between two morphologically and ecologically divergent species on Yakushima Island-the light-purple flowered Rhododendron eriocarpum native to seaside habitats and the red flowered R. indicum native to riverside habitats. By investigation of morphological traits and amplified fragment length polymorphisms (AFLP) we found that hybrid individuals morphologically and genetically intermediate between the two species grow mainly in the seaside areas of the island. The degree of morphological and genetic variation was different among the seaside hybrid populations. Although most pollinator species were exclusive to one of the two flower color groups, the halictine bees of Lasioglossum were observed in both the color types. The crucial interspecific incompatibility after pollination has not been previously described. Geographic distance between the populations is likely to be an important primary factor in bringing about natural hybridization and determining the degree of introgression between R. eriocarpum and R. indicum. The fact that hybrids occur mostly in the seaside area on Yakushima Island indicates that asymmetrical introgression occurs from R. indicum to R. eriocarpum. Alternatively, strong habitat-mediated selection from recurrent floods may prevent the hybrids from colonizing riverside habitats.

Natural hybridization origin of Rhododendron agastum (Ericaceae) in Yunnan, China: inferred from morphological and molecular evidence.

The natural hybridization that occurs between two sympatric species of Rhododendron subgenus Hymenanthes in Yunnan, China, was investigated. The assumed parents, Rhododendron delavayi Franch. and R. decorum Franch., are morphologically distinct, and the putative hybrid species, R. agastum Balf. f. et W. W. Smith, has an intermediate morphology. We used the main morphological characters, sequences of the nuclear ribosomal DNA ITS region, and the chloroplast DNA trnL-F intronspacer to analyze the three species, and compared these morphological and molecular data with an artificial hybrid between R. decorum (female symbol) x R. delavayi (male symbol). From the results, we conclude that R. agastum is a natural hybrid between a female R. delavayi and a male R. decorum.

[Pharmacognostic studies of Tibetan medicine da-li].

Da-Li is a traditional medicine of Tibetan, its original plant is Rhododendron primulaeflorum Bur. et Franch and R. anthopogonoides Maxim. This paper reports the identification of Flos et Folium Rhododendri Primulaeflori on its macroscopic character, microscopical charactersitic and TLC. The comparison between the Flos et Folium Rhododendri Primulaeflori and Flos et Folium Rhododendri anthopogonoidi is also reported.