Resolution, conflict and rate shifts: insights from a densely sampled plastome phylogeny for Rhododendron (Ericaceae).

BACKGROUND AND AIMS: Rhododendron is a species-rich and taxonomically challenging genus due to recent adaptive radiation and frequent hybridization. A well-resolved phylogenetic tree would help to understand the diverse history of Rhododendron in the Himalaya-Hengduan Mountains where the genus is most diverse. METHODS: We reconstructed the phylogeny based on plastid genomes with broad taxon sampling, covering 161 species representing all eight subgenera and all 12 sections, including ~45 % of the Rhododendron species native to the Himalaya-Hengduan Mountains. We compared this phylogeny with nuclear phylogenies to elucidate reticulate evolutionary events and clarify relationships at all levels within the genus. We also estimated the timing and diversification history of Rhododendron, especially the two species-rich subgenera Rhododendron and Hymenanthes that comprise >90 % of Rhododendron species in the Himalaya-Hengduan Mountains. KEY RESULTS: The full plastid dataset produced a well-resolved and supported phylogeny of Rhododendron. We identified 13 clades that were almost always monophyletic across all published phylogenies. The conflicts between nuclear and plastid phylogenies suggested strongly that reticulation events may have occurred in the deep lineage history of the genus. Within Rhododendron, subgenus Therorhodion diverged first at 56 Mya, then a burst of diversification occurred from 23.8 to 17.6 Mya, generating ten lineages among the component 12 clades of core Rhododendron. Diversification in subgenus Rhododendron accelerated c. 16.6 Mya and then became fairly continuous. Conversely, Hymenanthes diversification was slow at first, then accelerated very rapidly around 5 Mya. In the Himalaya-Hengduan Mountains, subgenus Rhododendron contained one major clade adapted to high altitudes and another to low altitudes, whereas most clades in Hymenanthes contained both low- and high-altitude species, indicating greater ecological plasticity during its diversification. CONCLUSIONS: The 13 clades proposed here may help to identify specific ancient hybridization events. This study will help to establish a stable and reliable taxonomic framework for Rhododendron, and provides insight into what drove its diversification and ecological adaption. Denser sampling of taxa, examining both organelle and nuclear genomes, is needed to better understand the divergence and diversification history of Rhododendron.

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.

Can species distribution models and molecular tools help unravel disjunct distribution of Rhododendron arboreum?

The apparent absence of Himalayan low-elevation taxa in the central Indian region and resumption of their distribution in the high elevation of Western Ghats has puzzled biogeographers for several decades. Many theories have been proposed to explain this but attempts remain futile owing to insufficient empirical support. Here, we have employed a montane tree species, Rhododendron arboreum to investigate this pattern by integrating past ecological niche modelling with molecular signatures. Reconstruction of paleo-ecological niche from interglacial to Last Glacial Maxima (LGM) portrayed a gradual depletion of vegetation cover with extreme impoverishment in the Holocene. A similar pattern was also reflected from genetic signatures; population history revealed a very recent split between the Himalayas and Western Ghats in the late Quaternary. A few other tree species exhibiting the same disjunction demonstrated a similar modification of paleo-ecological niche from last interglacial. The study clearly indicated that the populations in the Western Ghats to be a relictual remnants of a once continuous distribution of R. arboreum.

Comparative Analysis of Far East Sikhotinsky Rhododendron (Rh. sichotense) and East Siberian Rhododendron (Rh. adamsii) Using Supercritical CO2-Extraction and HPLC-ESI-MS/MS Spectrometry.

Rhododendron sichotense Pojark. and Rhododendron adamsii Rheder have been actively used in ethnomedicine in Mongolia, China and Buryatia (Russia) for centuries, as an antioxidant, immunomodulating, anti-inflammatory, vitality-restoring agent. These plants contain various phenolic compounds and fatty acids with valuable biological activity. Among green and selective extraction methods, supercritical carbon dioxide (SC-CO2) extraction has been shown to be the method of choice for the recovery of these naturally occurring compounds. Operative parameters and working conditions have been optimized by experimenting with different pressures (300-400 bar), temperatures (50-60 °C) and CO2 flow rates (50 mL/min) with 1% ethanol as co-solvent. The extraction time varied from 60 to 70 min. A HPLC-UV-VIS-ESI-MS/MS technique was applied to detect target analytes. A total of 48 different biologically active components have been identified in the Rh. adamsii SC-CO2 extracts. A total of 31 different biologically active components have been identified in the Rh. sichotense SC-CO2 extracts.

Genome survey sequencing and identification of genomic SSR markers for Rhododendron micranthum.

Rhododendron micranthum is an evergreen shrub species widely distributed in China that has high ornamental and medicinal value. However, there is a lack of molecular and genomic data for this plant, which severely restricts the development of its relevant research. The objective of the present study was to conduct a first genomic survey of R. micranthum and determine its whole-genome sequencing scheme. Next-generation sequencing (Illumina Hi-Seq Xten) was used to measure the genome size of R. micranthum, K-mer analysis were employed to investigate its genomic profile. Finally, we conducted bioinformatics methods to performed SSR (simple sequence repeat) prediction based on the genomic data. The genome size of R. micranthum was estimated to be 554.22 Mb. The heterozygosity ratio was 0.93%, and the sequence repeat ratio was calculated to be 49.17%. The clean reads of R. micranthum were assembled into 2281551 scaffolds with a N50 value of 916 bp. A total of 479724 SSR molecular markers were identified in the R. micranthum genome, and 871656 pairs of primers designed for application. Among of them, 100 primer pairs were validated, and 71 primer pairs were successfully amplified. In summary, the R. micranthum genome is complex with high heterozygosity and low repeated sequences. In future whole-genome research in R. micranthum, higher-depth '2+3' (Illumina+PacBio) sequencing may yield better assembly results.

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.

Phylogeographic analysis suggests two origins for the riparian azalea Rhododendron indicum (L.) Sweet.

Ecological speciation is an important factor in the diversification of plants. The distribution of the woody species Rhododendron indicum, which grows along rivers and is able to withstand water flow when rivers flood (i.e. it is a rheophyte), is disjunct, in contrast to the widespread distribution of its relative, Rhododendron kaempferi. This study aimed to elucidate the phylogenetic relationships between R. indicum and R. kaempferi and the evolutionary processes that gave rise to them. The sequences of three non-coding chloroplast DNA regions (total length 1977 bp) were obtained from 21 populations covering the ranges of the two species. In addition, genome-wide SNPs were genotyped from 20 populations using a genotyping by sequencing method. Leaf morphologies were measured for eight representative populations. Two chloroplast DNA haplotypes, which were detected in R. indicum, were shared between the two species. Genome-wide SNPs identified two lineages in R. indicum and these lineages did not constitute a monophyletic group. Each of these two lineages was related to geographically close populations of R. kaempferi. Leaf morphology, which is a characteristic feature in rheophytes, was not differentiated between the two lineages in R. indicum. The morphological similarity between the two heterogeneous lineages may be a result of parallel evolution from R. kaempferi or of introgressive hybridization between the species due to strong selective pressure imposed by flooding.

Influence of Light Intensity and Photoperiod on the Seed Germination of Four Rhododendron Species in Taiwan.

BACKGROUND AND OBJECTIVE: There are 15 native Rhododendron species in Taiwan, among which 11 species are endemic and compose 73% of these native species. Although researchers predominantly use cuttings to propagate Rhododendron shrubs, there are no studies on the seed germination of Rhododendron species. The objective of this study was to evaluate the seed germination of four Rhododendron species in Taiwan under different light intensities and photoperiods. MATERIALS AND METHODS: Two experiments on the seed germination percentage of R. breviperulatum, R. kanehirai, R. ovatum and R. simsii were conducted in this study. The first experiment was to identify the seed germination percentage of these four Rhododendron species using different light intensities (0, 700, 1400 and 3200 lux). The second experiment was to clarify the seed germination percentage of these four Rhododendron species using different photoperiods (0, 1, 4 and 16 h). All statistical analyses were performed using Statistical Package for the Social Science (SPSS12.0) for Windows software program. The data were analyzed using Tukey's multiple range test at the p<0.05 significance level. RESULTS: After 30 days, no seed germination occurred in darkness. The highest average seed germination percentages were all observed at 700 lux: R. breviperulatum (83.3%), R. kanehirai (68.9%), R. ovatum (85.6%) and R. simsii (92.2%). The highest average germination percentages of seeds were observed in R. breviperulatum at 16 h (83.3%), R. kanehirai at 1 h (60.0%), R. ovatum at 16 h (84.4%) and R. simsii at 16 h (85.6%). According to the results, these four Rhododendron species required light for germination. There were significant differences (p<0.05) in the seed germination of these four Rhododendron species for light intensity greater than 700 lux. Similar results were observed with photoperiods. CONCLUSION: The seed germination percentage of R. breviperulatum, R. ovatum and R. simsii increased with increasing photoperiod.

Decoupled leaf and root carbon economics is a key component in the ecological diversity and evolutionary divergence of deciduous and evergreen lineages of genus Rhododendron.

PREMISE: We explored trait-trait and trait-climate relationships for 27 Rhododendron species while accounting for phylogenetic relationships and within-species variation to investigate whether leaf and root traits are coordinated across environments and over evolutionary time, as part of a whole-plant economics spectrum. METHODS: We examined specific leaf area (SLA) and four root traits: specific root length (SRL), specific root tip abundance (SRTA), first order diameter, and link average length, for plants growing in a cold, seasonal climate (Kirtland, Ohio) and a warmer, less seasonal climate (Federal Way, Washington) in the United States. We estimated a phylogeny and species' climate of origin, determined phylogenetic signal on mean traits and within-species variation, and used phylogenetically informed analysis to compare trait-trait and trait-climate relationships for deciduous and evergreen lineages. RESULTS: Mean SLA and within-species variation in SRL were more similar between close relatives than expected by chance. SLA and root traits differed according to climate of origin and across growth environments, though SLA differed within- and among-species less than roots. A negative SRL-SRTA correlation indicates investment in foraging scale vs. precision as a fundamental trade-off defining the root economic spectrum. Also, the deciduous clade exhibited a strong negative relationship between SLA and SRL, while evergreen clades showed a weaker positive or no relationship. CONCLUSIONS: Our work suggests that natural selection has shaped relationships between above- and belowground traits in genus Rhododendron and that leaf and root traits may evolve independently. Morphological decoupling may help explain habitat diversity among Rhododendron species, as well as the changes accompanying the divergence of deciduous and evergreen lineages.

DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya-Hengduan Mountains.

The Himalaya-Hengduan Mountains encompass two global biodiversity hotspots with high levels of biodiversity and endemism. This area is one of the diversification centres of the genus Rhododendron, which is recognized as one of the most taxonomically challenging plant taxa due to recent adaptive radiations and rampant hybridization. In this study, four DNA barcodes were evaluated on 531 samples representing 173 species of seven sections of four subgenera in Rhododendron, with a high sampling density from the Himalaya-Hengduan Mountains employing three analytical methods. The varied approaches (nj, pwg and blast) had different species identification powers with blast performing best. With the pwg analysis, the discrimination rates for single barcodes varied from 12.21% to 25.19% with ITS < rbcL < matK < psbA-trnH. Combinations of ITS + psbA-trnH + matK and the four barcodes showed the highest discrimination ability (both 41.98%) among all possible combinations. As a single barcode, psbA-trnH performed best with a relatively high performance (25.19%). Overall, the three-marker combination of ITS + psbA-trnH + matK was found to be the best DNA barcode for identifying Rhododendron species. The relatively low discriminative efficiency of DNA barcoding in this genus (~42%) may possibly be attributable to too low sequence divergences as a result of a long generation time of Rhododendron and complex speciation patterns involving recent radiations and hybridizations. Taking the morphology, distribution range and habitat of the species into account, DNA barcoding provided additional information for species identification and delivered a preliminary assessment of biodiversity for the large genus Rhododendron in the biodiversity hotspots of the Himalaya-Hengduan Mountains.

[The analyses and identification of Flos rhododendri mollis and Flos chrysanthemi indici via infrared spectroscopy].

In this study, major chemical components of Flos rhododendri mollis and Flos chrysanthemi indici were characterized using Fourier transform infrared spectroscopy (FTIR). For Flos rhododendri mollis, the bands at 1,648 and 1,543 cm(-1) were attributed to amide I and amide II , respectively, indicating that it contained proteins probably resulting in immunization. In case of Flos chrysanthemi indici, stretching vibration of C==O function group was responsible for the bands at 1,734 and 1,515 cm(-1), as a result of essential oils, lipids, etc. Since FTIR spectra of Flos rhododendri mollis and Flos chrysanthemi indici are almost identical and it is difficult to discriminate them, two-step identification was investigated via secondary derivative of the FTIR spectra. The bands at 1,656 and 1,515 cm(-1) corresponds to flavonoides in Flos rhododendri mollis and Flos chrysanthemi indici. In the secondary derivative of the FTIR spectrum of Flos chrysanthemi indici, characteristic bands of inulin were present at 1,163, 1,077, 1,026, 986 and 869 cm(-1), and therefore Flos chrysanthemi indici contained inulin as well. Tri-step identification was carried out for Flos rhododendri mollis and Flos chrysanthemi indici by means of comparing their 2D-IR correlation spectra in different wave number range. In the characteristic range of flavonoides (1,700-1,400 cm(-1)), Flos rhododendri mollis exhibited 3 obvious autopeaks, while 10 autopeaks were visualized in the 2D-IR correlation spectrum of Flos chrysanthemi indici Moreover, in the characteristic range of glucoside (1,250-900 cm(-1)), 10 and 9 autopeaks were present in the 2D-IR correlation spectra of Flos rhododendri mollis and Flos chrysanthemi indici, respectively. Therefore, the tri-step identification of FTIR is a time-saving; accurate, cost-saving and convenient method to effectively distinguish traditional Chinese medicines.

[Distribution pattern of Rhododendron aganniphum var. schizopeplum population at the timberline of Sejila Mountains, Tibet of China].

Rhododendron aganniphum var. schizopeplum grows at the elevation of 4300-4500 m in Sejila Mountains, being a dominant species of shrub layer at the timberline of southeast Tibet. Based on the sampling plot investigation and basal diameter structure analysis, this paper studied the distribution pattern of R. aganniphum var. schizopeplum population at the timberline of Sejila Mountains. With the increase of altitude, the population density of R. aganniphum var. schizopeplum increased, but the population height decreased. At the same altitudes, the population density on the shady slope was larger than that on the sunny slope, while the basal diameter was smaller. On the two slope directions, the distribution of the basal diameter classes of the population appeared as "(see text for symbol)" shape, i. e., middle-aged individuals were more, while seedlings and saplings were relatively less. The intraspecific competition of R. aganniphum var. schizopeplum population was in the form of wave, being stronger on shady slope than on sunny slope, and existing self-thinning.

Bayesian analysis of biogeography when the number of areas is large.

Historical biogeography is increasingly studied from an explicitly statistical perspective, using stochastic models to describe the evolution of species range as a continuous-time Markov process of dispersal between and extinction within a set of discrete geographic areas. The main constraint of these methods is the computational limit on the number of areas that can be specified. We propose a Bayesian approach for inferring biogeographic history that extends the application of biogeographic models to the analysis of more realistic problems that involve a large number of areas. Our solution is based on a "data-augmentation" approach, in which we first populate the tree with a history of biogeographic events that is consistent with the observed species ranges at the tips of the tree. We then calculate the likelihood of a given history by adopting a mechanistic interpretation of the instantaneous-rate matrix, which specifies both the exponential waiting times between biogeographic events and the relative probabilities of each biogeographic change. We develop this approach in a Bayesian framework, marginalizing over all possible biogeographic histories using Markov chain Monte Carlo (MCMC). Besides dramatically increasing the number of areas that can be accommodated in a biogeographic analysis, our method allows the parameters of a given biogeographic model to be estimated and different biogeographic models to be objectively compared. Our approach is implemented in the program, BayArea.

Determination of genetic relationships between evergreen azalea cultivars in China using AFLP markers.

Evergreen azaleas are among the most important ornamental shrubs in China. Today, there are probably over 300 cultivars preserved in different nurseries, but with little information available on the cultivar itself or relationships between cultivars. Amplified fragment length polymorphism (AFLP) markers were employed to determine the genetic relationships between evergreen azalea cultivars in China. One hundred and thirty genotypes collected from gardens and nurseries, including cultivars classified in the groups East, West, Hairy, and Summer, unknown cultivars, and close species, were analyzed using three primer pairs. A total of 408 polymorphic fragments were generated by AFLP reactions with an average of 136 fragments per primer pair. The average values of expected heterozygosity and Shannon's information index were 0.3395 and 0.5153, respectively. Genetic similarities were generated based on Dice coefficients, used to construct a neighbor joining tree, and bootstrapped for 100 replicates in Treecon V1.3b. Principal coordinate analysis (PCO) was performed based on Dice distances using NTSYS-pc software. The AFLP technique was useful for analyzing genetic diversity in evergreen azaleas. Cluster analysis revealed that cultivars in the West and Summer groups were quite distinct from other groups in the four-group classification system and that the East and Hairy groups should be redefined.

Historical connectivity, contemporary isolation and local adaptation in a widespread but discontinuously distributed species endemic to Taiwan, Rhododendron oldhamii (Ericaceae).

Elucidation of the evolutionary processes that constrain or facilitate adaptive divergence is a central goal in evolutionary biology, especially in non-model organisms. We tested whether changes in dynamics of gene flow (historical vs contemporary) caused population isolation and examined local adaptation in response to environmental selective forces in fragmented Rhododendron oldhamii populations. Variation in 26 expressed sequence tag-simple sequence repeat loci from 18 populations in Taiwan was investigated by examining patterns of genetic diversity, inbreeding, geographic structure, recent bottlenecks, and historical and contemporary gene flow. Selection associated with environmental variables was also examined. Bayesian clustering analysis revealed four regional population groups of north, central, south and southeast with significant genetic differentiation. Historical bottlenecks beginning 9168-13,092 years ago and ending 1584-3504 years ago were revealed by estimates using approximate Bayesian computation for all four regional samples analyzed. Recent migration within and across geographic regions was limited. However, major dispersal sources were found within geographic regions. Altitudinal clines of allelic frequencies of environmentally associated positively selected outliers were found, indicating adaptive divergence. Our results point to a transition from historical population connectivity toward contemporary population isolation and divergence on a regional scale. Spatial and temporal dispersal differences may have resulted in regional population divergence and local adaptation associated with environmental variables, which may have played roles as selective forces at a regional scale.

Paternity determination of interspecific rhododendron hybrids by genomic in situ hybridization (GISH).

Genomic in situ hybridization (GISH) has been proved to be the most effective and accurate technique for confirmation of hybrid character. The objective of our study was to adapt and optimize a GISH protocol for identification of donor chromatin in hybrids obtained by interspecific crosses between five Rhododendron taxa (R. aureum, R. brachycarpum, R. catawbiense 'Catharine van Tol', R. catawbiense 'Nova Zembla', and R. yakushimanum 'Koichiro Wada'). Positive results were obtained only when we used mitotic chromosome spreads prepared from anthers. The best differentiation of maternal and paternal chromosomes in hybrid genomes was obtained when 50 ng of probe was applied together with blocking DNA at a concentration of 3.0 microg/microL. The results demonstrate that GISH is a practical tool for detection of alien genomes and analysis of the constitution of the chromosomes in rhododendron hybrids.

[Differentiation of Rhododendron based on the infrared spectra of petals].

Several techniques were used to identify and classify plants. Mid-infrared spectroscopy combined with appropriate software was used in an attempt to differentiate different subgenus from Rhododendron. Fourier transform infrared (FTIR) spectroscopy was used for obtaining vibrational spectra of 46 petals from Rhododendron. Very minor differences were observed in the FTIR spectra among four subgenuses. For the purpose of rapid differentiation, libraries of spectra were created using samples from each subgenus variety. Spectra of unknown samples were recorded and compared with those of the libraries and the rate of affinity (the match value) was measured automatically using the appropriate software (OMNIC). The results showed that petal samples from different subgenus varieties can be differentiated from each other. The study demonstrates that combining FTIR spectroscopy with appropriate analysis method can classify Rhododendron plants at subgenus level. It offers a potential method for the taxonomic research on plants system.

Azalea phylogeny reconstructed by means of molecular techniques.

Plants belonging to the Rhododendron subgenera Pentanthera (deciduous) and Tsutsusi and Azaleastrum (evergreen) are called azaleas. Concerning their mutual phylogenetic positions, the Pentanthera subgenus is closer to evergreen rhododendrons (subgenera Rhododendron and Hymenanthes) than to the Tsutsusi subgenus. Both azalea types are important ornamentals with a long breeding tradition. Different hybrid groups are often named after the supposed principal ancestor species. Molecular techniques for phylogenetic and kinship research have been evaluated to a great extent. First, some studies using comparative gene sequencing are presented; this approach was then widened to the use of molecular markers to reveal more detailed genetic relationships. Finally, the use of candidate genes as functional markers for the assessment of genetic diversity is presented. This opens new research lines to the genetic mapping of plant traits and azalea genomics.

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.

Testate amoebae (Arcellinida and Euglyphida) vs. Ericoid mycorrhizal and DSE fungi: a possible novel interaction in the mycorrhizosphere of ericaceous plants?

Common occurrence of testate amoebae (TA) in the rhizosphere of mycorrhizal plants indicates existence of yet undocumented ecological interactions, involving three distinct groups of organisms: soil protists, mycorrhizal fungi, and their host plants. This tripartite relationship was to date investigated only to a limited extent, despite its probable importance for processes taking place in the mycorrhizosphere. In this study, we (1) explored spectra of different TA genera naturally associated with the rhizoplane of three autochthonous European Rhododendron species, (2) screened natural fungal colonization of the TA shells occupying the rhizoplane of selected rhododendrons, and (3) carried out two in vitro experiments addressing the question whether TA shells may serve as a nutrient source for ericoid mycorrhizal fungi (ErMF) and dark septate endophytes (DSE). Our field observations indicated that TA regularly associated with the rhizoplane of all screened rhododendrons and that ErMF and/or DSE associated with their roots possibly exploited the TA shells as a nutrient source. We were unable to detect any major differences among the TA spectra from the rhizoplanes with respect to the three Rhododendron species. The spectra were dominated by Diplochlamys, Centropyxis, Cyclopyxis, Euglypha, Trinema, and Assulina. Positive, neutral, and negative associations were found for various TA genera x Rhododendron species combinations. The highest fungal colonization was observed in Centropyxidae and Trigonopyxidae, reaching up to 45% of the shells in the case of Trigonopyxis. In the in vitro experiments, both ErMF Rhizoscyphus ericae and DSE Phialocephala fortinii regularly colonized TA shells, utilizing them as a source of nutrients. We hypothesize a complex relationship between ErMF-DSE and TA. If corroborated, it would represent an interesting nutrient loop in the mycorrhizosphere of ericaceous plants.