Search

Interrogating the ecological and geographic factors that influence population divergence dynamics can reveal why some groups of organisms diversify more prolifically than others. One such group is the heathers (Erica, Ericaceae), the largest plant genus in the Cape Floristic Region. We study Erica abietina, a highly variable species complex with four subspecies differing in geographic range, habitat and pollination syndrome. We test for population differentiation, hybridisation, introgression and pollinator-driven divergence using genotyping-by-sequencing on samples across the entire distribution. We find five variably distinct genetic groups, with one subspecies comprising two independent lineages that are geographically isolated and occur on different soil types. Phylogenetic analysis suggests two independent shifts between bird and insect pollination, with accompanying genetic divergence. However, for one pair of populations with different pollinators, we uncover several individuals of hybrid origin at a site of sympatry. These results suggest that floral differentiation driven by divergent selection acts in concert with geographic isolation to maintain reproductive isolation and promote speciation. Our investigations reveal a highly dynamic system whose diversity has been shaped by a variety of interacting forces. We suggest that such a system could be a model for much of the diversification of the Cape flora., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Fully mycoheterotrophic plants can be difficult to place in plant phylogeny due to elevated substitution rates associated with photosynthesis loss. This potentially limits the effectiveness of downstream analyses of mycoheterotrophy that depend on accurate phylogenetic inference. Although mitochondrial genomic data sets are rarely used in plant phylogenetics, theory predicts that they should be resilient to long-branch artefacts, thanks to their generally slow evolution, coupled with limited rate elevation in heterotrophs. We examined the utility of mitochondrial genomes for resolving contentious higher-order placements of mycoheterotrophic lineages in two test cases: monocots (focusing on Dioscoreales) and Ericaceae. We find Thismiaceae to be distantly related to Burmanniaceae in the monocot order Dioscoreales, conflicting with current classification schemes based on few gene data sets. We confirm that the unusual Afrothismia is related to Taccaceae-Thismiaceae, with a corresponding independent loss of photosynthesis. In Ericaceae we recovered the first well supported relationships among its five major lineages: mycoheterotrophic Ericaceae are not monophyletic, as pyroloids are inferred to be sister to core Ericaceae, and monotropoids to arbutoids. Genes recovered from mitochondrial genomes collectively resolved previously ambiguous mycoheterotroph higher-order relationships. We propose that mitochondrial genomic data should be considered in standardised gene panels for inferring overall plant phylogeny., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Background: Rhododendron molle (Ericaceae) is a traditional Chinese medicine, which has been used to treat rheumatism and relieve pain since ancient times. The characteristic grayanoids of this plant have been demonstrated to be the chemical basis for the analgesic activity. Moreover, unlike morphine, these diterpenoids are non-addictive. Grayanoids mainly distribute in the leaves, flowers, roots, and fruits of R. molle, with low content. Currently the research on the biosynthesis of grayanoids is hindered, partially due to lack of the genomic information., Results: In the present study, a total of 744 Mb sequences were generated and assembled into 13 chromosomes. An ancient whole-genome duplication event (Ad-β) was discovered that occurred around 70 million years ago. Tandem and segmental gene duplications led to specific gene expansions in the terpene synthase and cytochrome P450 (CYP450) gene families. Two diterpene synthases were demonstrated to be responsible for the biosynthesis of 16α-hydroxy-ent-kaurane, the key precursor for grayanoids. Phylogenetic analysis revealed a species-specific bloom of the CYP71AU subfamily, which may involve the candidate CYP450s responsible for the biosynthesis of grayanoids. Additionally, three putative terpene biosynthetic gene clusters were found., Conclusions: We reported the first genome assembly of R. molle and investigated the molecular basis underpinning terpenoids biosynthesis. Our work provides a foundation for elucidating the complete biosynthetic pathway of grayanoids and studying the terpenoids diversity in R. molle., (© 2022. The Author(s).)

Cranberry (Vaccinium macrocarpon) is a member of the Heath family (Ericaceae) and is a temperate low-growing woody perennial native to North America that is both economically important and has significant health benefits. While some native varieties are still grown today, breeding programs over the past 50 years have made significant contributions to improving disease resistance, fruit quality and yield. An initial genome sequence of an inbred line of the wild selection 'Ben Lear,' which is parent to multiple breeding programs, provided insight into the gene repertoire as well as a platform for molecular breeding. Recent breeding efforts have focused on leveraging the circumboreal V. oxycoccos, which forms interspecific hybrids with V. macrocarpon, offering to bring in novel fruit chemistry and other desirable traits. Here we present an updated, chromosome-resolved V. macrocarpon reference genome, and compare it to a high-quality draft genome of V. oxycoccos. Leveraging the chromosome resolved cranberry reference genome, we confirmed that the Ericaceae has undergone two whole genome duplications that are shared with blueberry and rhododendron. Leveraging resequencing data for 'Ben Lear' inbred lines, as well as several wild and elite selections, we identified common regions that are targets of improvement. These same syntenic regions in V. oxycoccos, were identified and represent environmental response and plant architecture genes. These data provide insight into early genomic selection in the domestication of a native North American berry crop., Competing Interests: NO authors have competing interests.

Rhododendron purdomii (Ericaceae) is an endangered ornamental species endemic to the Qinling Mountains of China. Due to the impact of climate change and human disturbance, R. purdomii is threatened by habitat loss, and conservation of this species is urgently needed. In this study, we developed and characterized 13 novel microsatellite markers for R. purdomii based on next-generation sequencing data. For the 13 microsatellite markers in three R. purdomii populations, the number of alleles ranged from two to 12, the number of effective alleles was from 1.000 to 8.892, Shannon's information index was from 0.000 to 2.320, and the observed and expected heterozygosity were from 0.000 to 1.000 and from 0.000 to 0.888, respectively. Private alleles were found in all three populations. Moderate differentiation between population pairs was indicated by pairwise F ST values. The microsatellite markers developed in this study will provide opportunities for examining the genetic diversity and population structure of R. purdomii and contribute to the effective conservation of this species.

Background: The coincidence of long distance dispersal (LDD) and biome shift is assumed to be the result of a multifaceted interplay between geographical distance and ecological suitability of source and sink areas. Here, we test the influence of these factors on the dispersal history of the flowering plant genus Erica (Ericaceae) across the Afrotemperate. We quantify similarity of Erica climate niches per biogeographic area using direct observations of species, and test various colonisation scenarios while estimating ancestral areas for the Erica clade using parametric biogeographic model testing., Results: We infer that the overall dispersal history of Erica across the Afrotemperate is the result of infrequent colonisation limited by geographic proximity and niche similarity. However, the Drakensberg Mountains represent a colonisation sink, rather than acting as a "stepping stone" between more distant and ecologically dissimilar Cape and Tropical African regions. Strikingly, the most dramatic examples of species radiations in Erica were the result of single unique dispersals over longer distances between ecologically dissimilar areas, contradicting the rule of phylogenetic biome conservatism., Conclusions: These results highlight the roles of geographical and ecological distance in limiting LDD, but also the importance of rare biome shifts, in which a unique dispersal event fuels evolutionary radiation.

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background and Aims: Flowering plants show a high diversity of pollen morphology, assumed to reflect not only variations in the underlying design, but also stress imposed by ecological conditions related to pollen survival and germination. Both components are expected to constrain the accumulation of pollen disparity. However, this assumption has rarely been tested using empirical data., Methods: This study is designed to test this hypothesis by inferring the accumulation of pollen disparity in Ericaceae, a large eudicot family with recent, ongoing radiations, with focus on three functionally significant pollen characters using a dated phylogeny., Key Results: Multiple lines of evidence supported the hypothesis that pollen disparity in Ericaceae did not evolve steadily but rather pulsed over time, clearly decoupling from the relative constant rate pattern of species diversification inferred. In a 3-D pollen morphospace, most major clades appear to occupy distinct neighbouring regions, whereas the subfamily Epacridoideae overlaps extensively with other subfamilies. No evidence for correlations was found between dimension of pollen disparity and species diversity at either the subfamily or generic level. Furthermore, the distribution of species in present pollen morphospace showed a strong central tendency, with the core compartment containing a large number of species from species-rich genera., Conclusions: The recovered evidence fits well with the expectations of limitations on available pollen morphological disparity, and suggests that innovation of pollen germination traits may have little effect on species diversification., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Specialist herbivores have often evolved highly sophisticated mechanisms to counteract defenses mediated by major plant secondary-metabolites. Plant species of the herbivore host range often display high chemical diversity and it is not well understood how specialist herbivores respond to this chemical diversity. Pieris larvae overcome toxic products from glucosinolate hydrolysis, the major chemical defense of their Brassicaceae hosts, by expressing nitrile-specifier proteins (NSP) in their gut. Furthermore, Pieris butterflies possess so-called major allergen (MA) proteins, which are multi-domain variants of a single domain major allergen (SDMA) protein expressed in the guts of Lepidopteran larvae. Here we show that Pieris larvae fine-tune NSP and MA gene expression depending on the glucosinolate profiles of their Brassicaceae hosts. Although the role of MA is not yet fully understood, the expression levels of NSP and MA in larvae that fed on plants whose glucosinolate composition varied was dramatically changed, whereas levels of SDMA expression remained unchanged. In addition, we found a similar regulation pattern among these genes in larvae feeding on Arabidopsis mutants with different glucosinolate profiles. Our results demonstrate that Pieris larvae appear to use different host plant adaptive genes to overcome a wide range of glucosinolate profiles in their host plants.

Background: Vaccinium uliginosum (Ericaceae) is an important wild berry having high economic value. The white-fruited V. uliginosum variety found in the wild lacks anthocyanin and bears silvery white fruits. Hence, it is a good resource for investigating the mechanism of fruit color development. This study aimed to verify the differences in the expression levels of some structural genes and transcription factors affecting the anthocyanin biosynthesis pathway by conducting high-throughput transcriptome sequencing and real-time PCR analysis by using the ripening fruits of V. uliginosum and the white-fruited variety., Results: We annotated 42,837 unigenes. Of the 325 differentially expressed genes, 41 were up-regulated and 284 were down-regulated. Further, 11 structural genes of the flavonoid pathway were up-regulated, whereas two were down-regulated. Of the seven genes encoding transcription factors, five were up-regulated and two were down-regulated. The structural genes VuCHS, VuF3'H, VuFHT, VuDFR, VuANS, VuANR, and VuUFGT and the transcription factors VubHLH92, VuMYB6, VuMYBPA1, VuMYB11, and VuMYB12 were significantly down-regulated. However, the expression of only VuMYB6 and VuMYBPA1 rapidly increased during the last two stages of V. uliginosum when the fruit was ripening, consistent with anthocyanin accumulation., Conclusions: VuMYB6 was annotated as MYB1 by the BLAST tool. Thus, the white fruit color in the V. uliginosum variant can be attributed to the down-regulation of transcription factors VuMYB1 and VuMYBPA1, which leads to the down-regulation of structural genes associated with the anthocyanin synthesis pathway.

Premise of the Study: Phylogenomic studies employing large numbers of genes, including those based on plastid genomes (plastomes), are becoming common. Nonphotosynthetic plants such as mycoheterotrophs (which rely on root-associated fungi for essential nutrients, including carbon) tend to have highly elevated rates of plastome evolution, substantial genome reduction, or both. Mycoheterotroph plastomes therefore provide excellent test cases for investigating how extreme conditions impact phylogenomic inference., Methods: We used parsimony and likelihood analysis of protein-coding gene sets from published and newly completed plastomes to infer the phylogenetic placement of taxa from the 10 angiosperm families in which mycoheterotrophy evolved., Key Results: Despite multiple very long branches that reflect elevated substitution rates, and frequently patchy gene recovery due to genome reduction, inferred phylogenetic placements of most mycoheterotrophic lineages in DNA-based likelihood analyses are both well supported and congruent with other studies. Amino-acid-based likelihood placements are broadly consistent with DNA-based inferences, but extremely rate-elevated taxa can have unexpected placements-albeit with weak support. In contrast, parsimony analysis is strongly misled by long-branch attraction among many distantly related mycoheterotrophic monocots., Conclusions: Mycoheterotrophic plastomes provide challenging cases for phylogenomic inference, as substitutional rates can be elevated and genome reduction can lead to sparse gene recovery. Nonetheless, diverse likelihood frameworks provide generally well-supported and mutually concordant phylogenetic placements of mycoheterotrophs, consistent with recent phylogenetic studies and angiosperm-wide classifications. Previous predictions of parallel photosynthesis loss within families are supported for Burmanniaceae, Ericaceae, Gentianaceae, and Orchidaceae. Burmanniaceae and Thismiaceae should not be combined as a single family in Dioscoreales., (© 2018 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America.)

The circumarctic ranges of arctic-alpine plants are thought to have been established in the late Pliocene/early Pleistocene, when the modern arctic tundra was formed in response to climate cooling. Previous findings of range-wide genetic structure in arctic-alpine plants have been thought to support this hypothesis, but few studies have explicitly addressed the temporal framework of the genetic structure. Here, we estimated the demographic history of the genetic structure in the circumarctic Kalmia procumbens using sequences of multiple nuclear loci and examined whether its genetic structure reflects prolonged isolation throughout the Pleistocene. Both Bayesian clustering and phylogenetic analyses revealed genetic distinction between alpine and arctic regions, whereas detailed groupings were somewhat discordant between the analyses. By assuming a population grouping based on the phylogenetic analyses, which likely reflects a deeper intraspecific divergence, we conducted model-based analyses and demonstrated that the intraspecific genetic divergence in K. procumbens likely originated during the last glacial period. Thus, there is no need to postulate range separation throughout the Pleistocene to explain the current genetic structure in this species. This study demonstrates that range-wide genetic structure in arctic-alpine plants does not necessarily result from the late Pliocene/early Pleistocene origin of their circumarctic ranges and emphasizes the importance of a temporal framework of the current genetic structure for understanding the biogeographic history of the arctic flora., (© 2017 John Wiley & Sons Ltd.)

Ericaceae (the heather family) is a large and diverse group of plants that forms elaborate symbiotic relationships with mycorrhizal fungi, and includes several nonphotosynthetic lineages. Using an extensive sample of fully mycoheterotrophic (MH) species, we explored inter- and intraspecific variation as well as selective constraints acting on the plastomes of these unusual plants. The plastomes of seven MH genera were analysed in a phylogenetic context with two geographically disparate individuals sequenced for Allotropa, Monotropa, and Pityopus. The plastomes of nonphotosynthetic Ericaceae are highly reduced in size (c. 33-41 kbp) and content, having lost all photosynthesis-related genes, and are reduced to encoding housekeeping genes as well as a protease subunit (clpP)-like and acetyl-CoA carboxylase subunit D (accD)-like open reading frames. Despite an increase in the rate of their nucleotide substitutions, the remaining protein-coding genes are typically under purifying selection in full MHs. We also identified ribosomal proteins under relaxed or neutral selection. These plastomes also exhibit striking structural rearrangements. Intraspecific variation within MH Ericaceae ranges from a few differences (Allotropa) to extensive population divergences (Monotropa, Hypopitys), which indicates that cryptic speciation may be occurring in several lineages. The pattern of gene loss within fully MH Ericaceae plastomes suggests an advanced state of degradation., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

The precise spatial-temporal coordination of cell division and differentiation is necessary for the correct formation of tissues, organs, and the organism as a whole. This coordination has been implemented by the intercellular communication mediated by signaling molecules and receptors that selectively recognize them. Membrane receptor kinases of ERECTA family regulate inflorescence and flower structure, the formation of root epidermis and adaptation responses. The characterization of the ERECTA genes of flowering plant pinesap Monotropa hypopitys with unique development features can enrich the knowledge about the kinase ERECTA functions and conserved development processes with their participation. Transcriptomic and genomic search with the subsequent structural-phylogenetic analysis identified the mRNA of a gene of serine-threonine kinase receptor with leucine-rich repeats of MhyERL1, which is the only ortholog of the ERECTA family kinases of pinesap. A quantitative analysis of the MhyERL1 gene transcripts has revealed its expression in all analyzed pinesap tissues with maximum levels in the flowers. MhyERL1 is probably involved in defining the inflorescence and flower architecture, and the formation of the pinesap root epidermis. The cascades involving ERL1 are apparently conserved. The exception are pathways associated with the development of above-ground vegetative structures, and the immune response to fungal pathogens probably lost in the process of the pinesap adaptation to unfavorable environmental conditions.

The gene encoding the transcription factor LEAFY was identified in the genome of the mycoheterotrophic plant, pinesap Monotropa hypopitys. In the transcriptomes of roots, bracts, and flowers of flowering pinesaps, the MhyLFY gene expression was absent. These data suggest the conservativeness of the LFY-dependent mechanism of flower meristem identity and flower formation in heterotrophic species with some differences associated to the specificity of development and the structure of such plants. The pinesap flowering under the control of the transcription factor MhyLFY may be initiated either in an embryonic inflorescence during spring dormancy release of adventitious root buds or in an inflorescence of a growing reproductive stem after photoperiodic induction.

Mediterranean forests are fragile ecosystems vulnerable to recent global warming and reduction of precipitation, and a long-term negative effect is expected on vegetation with increasing drought and in areas burnt by fires. We investigated the spatial distribution of genetic variation of Arbutus unedo in the western Iberia Peninsula, using plastid markers with conservation and provenance regions design purposes. This species is currently undergoing an intense domestication process in the region, and, like other species, is increasingly under the threat from climate change, habitat fragmentation and wildfires. We sampled 451 trees from 15 natural populations from different ecological conditions spanning the whole species' distribution range in the region. We applied Bayesian analysis and identified four clusters (north, centre, south, and a single-population cluster). Hierarchical AMOVA showed higher differentiation among clusters than among populations within clusters. The relatively low within-clusters differentiation can be explained by a common postglacial history of nearby populations. The genetic structure found, supported by the few available palaeobotanical records, cannot exclude the hypothesis of two independent A. unedo refugia in western Iberia Peninsula during the Last Glacial Maximum. Based on the results we recommend a conservation strategy by selecting populations for conservation based on their allelic richness and diversity and careful seed transfer consistent with current species' genetic structure.

Genes encoding six chitinases, five of which belong to classes I (MhCHI3 and MhCHI4), IV (MhCHI1), V (MhCHI5), and VII (MhCHI2), were identified in the transcriptome of the parasitic mixoheterotrophic plant Monotropa hypopitys. The transcription level of MhCHI5 and MhCHI1 was low; however, in the leaves (bracts) and roots it was higher than in flowers. MhCHI4 transcripts were detected primarily in the flowers and were almost absent in the roots, whereas the expression level of MhCHI3 was relatively high in all organs but maximum in the leaves (bracts).

Premise of the Study: Vegetative reproduction and spread through clonal growth plays an important role in arctic-alpine ecosystems with short cool growing seasons. Local variation in winter snow accumulation leads to discrete habitat types that may provide divergent conditions for sexual and vegetative reproduction. Therefore, we studied variation in clonal structure of a dominant, evergreen, dwarf shrub (Empetrum nigrum s.l. with the two taxa E. nigrum L. s.s. and E. hermaphroditum Hagerup) along a snow cover gradient and compared clonal diversity and spatial genetic structure between habitats., Methods: We studied 374 individual shoots using 105 polymorphic AFLP markers and analyses based on hierarchical clustering, clonal diversity indices, and small-scale spatial genetic structure with pairwise kinship coefficient. We used two approaches to define a threshold of genotypic distance between two samples that are considered the same clone. Clonality was examined among three habitats (exposed ridges, sheltered depressions, birch forest) differing in snow conditions replicated in four study regions in Norway and Sweden., Key Results: Clonality of E. hermaphroditum differed between habitats with an increase in clonal diversity with decreasing snow depth. Small-scale spatial genetic structure increased with decreasing clonal diversity and increasing clone size. In three study regions, E. hermaphroditum was the only species, whereas in one region E. nigrum also occurred, largely confined to exposed ridges., Conclusions: Our results demonstrated that snow cover in conjunction with associated habitat conditions plays an important role for the mode of propagation of the dwarf shrub E. hermaphroditum., (© 2016 Botanical Society of America.)

Background: Arbutus unedo is a valuable Mediterranean shrub as an ornamental plant as well as fruit tree. Fresh fruits of A. unedo are a good source of antioxidants, of vitamins C, E and carotenoids and also are characterized by the high content of mineral elements., Materials and Methods: The effects of gibberellic acid (GA 3 ) and cold stratification (CS) on seed germination performance were investigated in A. unedo seeds collected from three provenances in the Northern part of Greece. Seeds of each provenance were soaked in solutions of GA 3 (500, 1000 or 2000 ppm) for 24 h and subsequently were subjected to CS at 3 - 5°C for 0, 1, 2, and 3 months., Results: Non-stratified seeds of the three A. unedo provenances which were not treated with GA 3 solutions exhibited very low germination. However, seed germination was significantly improved after a one-month period of CS. Similarly, the non-stratified seeds of all three provenances became non-dormant after the treatment with 2000 ppm GA 3 and they germinated at high percentages. However, in untreated seeds with GA3, after a one-month CS period the seeds of the Pieria provenance exhibited higher germination percentage than that of Rodopi provenance seeds. Furthermore, in non-stratified seeds, the Pieria provenance seeds treated with GA3 germinated at higher percentages and more rapidly than those of the other two provenances., Conclusion: The results indicated that untreated seeds exhibited very low germination at 20/25°C. However, in all three provenances seed germinability was significantly improved by a one-month period of CS or treatment of seeds with 2000 ppm GA3. Furthermore, there was a considerable variability among seed provenances in response to the treatments which were applied.

Background: Chloroplasts of most plants are responsible for photosynthesis and contain a conserved set of about 110 genes that encode components of housekeeping gene expression machinery and photosynthesis-related functions. Heterotrophic plants obtaining nutrients from other organisms and their plastid genomes represent model systems in which to study the effects of relaxed selective pressure on photosynthetic function. The most evident is a reduction in the size and gene content of the plastome, which correlates with the loss of genes encoding photosynthetic machinery which become unnecessary. Transition to a non-photosynthetic lifestyle is expected also to relax the selective pressure on photosynthetic machinery in the nuclear genome, however, the corresponding changes are less known., Results: Here we report the complete sequence of the plastid genome of Monotropa hypopitys, an achlorophyllous obligately mycoheterotrophic plant belonging to the family Ericaceae. The plastome of M. hypopitys is greatly reduced in size (35,336 bp) and lacks the typical quadripartite structure with two single-copy regions and an inverted repeat. Only 45 genes remained presumably intact- those encoding ribosomal proteins, ribosomal and transfer RNA and housekeeping genes infA, matK, accD and clpP. The clpP and accD genes probably remain functional, although their sequences are highly diverged. The sets of genes for ribosomal protein and transfer RNA are incomplete relative to chloroplasts of a photosynthetic plant. Comparison of the plastid genomes of two subspecies-level isolates of M. hypopitys revealed major structural rearrangements associated with repeat-driven recombination and the presence of isolate-specific tRNA genes. Analysis of the M. hypopitys transcriptome by RNA-Seq showed the absence of expression of nuclear-encoded components of photosystem I and II reaction center proteins, components of cytochrome b6f complex, ATP synthase, ribulose bisphosphate carboxylase components, as well as chlorophyll from protoporphyrin IX biosynthesis pathway., Conclusions: With the complete loss of genes related to photosynthesis, NADH dehydrogenase, plastid-encoded RNA polymerase and ATP synthase, the M. hypopitys plastid genome is among the most functionally reduced ones characteristic of obligate non-photosynthetic parasitic species. Analysis of the M. hypopitys transcriptome revealed coordinated evolution of the nuclear and plastome genomes and the loss of photosynthesis-related functions in both genomes.

Background: The disproportionate species richness of the world's biodiversity hotspots could be explained by low extinction (the evolutionary "museum") and/or high speciation (the "hot-bed") models. We test these models using the largest of the species rich plant groups that characterise the botanically diverse Cape Floristic Region (CFR): the genus Erica L. We generate a novel phylogenetic hypothesis informed by nuclear and plastid DNA sequences of c. 60 % of the c. 800 Erica species (of which 690 are endemic to the CFR), and use this to estimate clade ages (using RELTIME; BEAST), net diversification rates (GEIGER), and shifts in rates of diversification in different areas (BAMM; MuSSE)., Results: The diversity of Erica species in the CFR is the result of a single radiation within the last c. 15 million years. Compared to ancestral lineages in the Palearctic, the rate of speciation accelerated across Africa and Madagascar, with a further burst of speciation within the CFR that also exceeds the net diversification rates of other Cape clades., Conclusions: Erica exemplifies the "hotbed" model of assemblage through recent speciation, implying that with the advent of the modern Cape a multitude of new niches opened and were successively occupied through local species diversification.

Although plastid genomes of flowering plants are typically highly conserved regarding their size, gene content and order, there are some exceptions. Ericaceae, a large and diverse family of flowering plants, warrants special attention within the context of plastid genome evolution because it includes both non-photosynthetic and photosynthetic species with rearranged plastomes and putative losses of "essential" genes. We characterized plastid genomes of three species of Ericaceae, non-photosynthetic Monotropa uniflora and Hypopitys monotropa and photosynthetic Pyrola rotundifolia, using high-throughput sequencing. As expected for non-photosynthetic plants, M. uniflora and H. monotropa have small plastid genomes (46 kb and 35 kb, respectively) lacking genes related to photosynthesis, whereas P. rotundifolia has a larger genome (169 kb) with a gene set similar to other photosynthetic plants. The examined genomes contain an unusually high number of repeats and translocations. Comparative analysis of the expanded set of Ericaceae plastomes suggests that the genes clpP and accD that are present in the plastid genomes of almost all plants have not been lost in this family (as was previously thought) but rather persist in these genomes in unusual forms. Also we found a new gene in P. rotundifolia that emerged as a result of duplication of rps4 gene.

Myco-heterotroph Monotropa hypopitys is a widely spread perennial herb used to study symbiotic interactions and physiological mechanisms underlying the development of non-photosynthetic plant. Here, we performed, for the first time, transcriptome-wide characterization of M. hypopitys miRNA profile using high throughput Illumina sequencing. As a result of small RNA library sequencing and bioinformatic analysis, we identified 55 members belonging to 40 families of known miRNAs and 17 putative novel miRNAs unique for M. hypopitys. Computational screening revealed 206 potential mRNA targets for known miRNAs and 31 potential mRNA targets for novel miRNAs. The predicted target genes were described in Gene Ontology terms and were found to be involved in a broad range of metabolic and regulatory pathways. The identification of novel M. hypopitys-specific miRNAs, some with few target genes and low abundances, suggests their recent evolutionary origin and participation in highly specialized regulatory mechanisms fundamental for non-photosynthetic biology of M. hypopitys. This global analysis of miRNAs and their potential targets in M. hypopitys provides a framework for further investigation of miRNA role in the evolution and establishment of non-photosynthetic myco-heterotrophs.

Arctic-alpine plants have enormous ranges in the Northern Hemisphere. Phylogeographic studies have provided insights into their glacial survival as well as their postglacial colonization history. However, our understanding of the population dynamics of disjunct alpine populations in temperate regions remains limited. During Pleistocene cold periods, alpine populations of arctic-alpine species in East Asia were either connected to an ice-free Beringia refugium or they persisted with prolonged isolation after their establishment. To estimate which of these scenarios is more likely, we elucidated the genetic structure of Phyllodoce caerulea (Ericaceae) in Beringia and northern Japan, East Asia. Sequence variation in multiple nuclear loci revealed that P. caerulea can be distinguished into northern and southern groups. A demographic analysis demonstrated that the north-south divergence did not predate the last glacial period and detected introgression from Phyllodoce aleutica, relative widely distributed in East Asia, exclusively into the southern group. Therefore, although there has been genetic divergence between northern Japan and Beringia in P. caerulea, the divergence is unlikely to have resulted from their prolonged geographic separation throughout several cycles of glacial and interglacial periods. Instead, our study suggests that the introgression contributed to the genetic divergence of P. caerulea and that the range of P. caerulea was plausibly connected between northern Japan and Beringia during the last glacial period. Overall, our study not only provides a biogeographic insight into alpine populations of arctic-alpine plants in East Asia but also emphasizes the importance of careful interpretation of genetic structure for inferring phylogeographic history.

Many arctic-alpine plants display a highly disjunct distribution between the Arctic/Boreal regions and the southern Asian mountains. Two main hypotheses have been proposed to explain the origin of this biogeographic pattern: (1) south-to-north migration in the late Pliocene/early Pleistocene, and (2) north-to-south migration during the Miocene. The genus Cassiope is disjunctly distributed between the Arctic/Boreal regions and the Himalayan-Hengduan Mountains (HHM) and was selected to test these hypotheses. We constructed a fossil-calibrated phylogeny of Ericaceae using two plastid regions to estimate the crown group age of Cassiope, and used sequence data from thousands of loci produced by restriction site associated DNA sequencing (RAD-seq) to reconstruct the phylogeny of Cassiope. We also performed Bayesian divergence time analysis and biogeographic analysis. The Cassiope crown group was estimated to have originated in the Miocene, which predates the onset of Northern hemisphere glaciation. All HHM species formed a clade together with one eastern Siberian species, and this clade was sister to all other Arctic/Boreal species. This topology implies a northern origin of Cassiope, which is confirmed by our biogeographic analysis. Our results thus suggest that the ancient north-to-south migration hypothesis is most consistent with the origin of Cassiope., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Intraspecific sequence polymorphism in the ITS region of the nuclear ribosomal operon was examined in ten populations of the parasitic plant Monotropa hypopitys from Belgorod oblast, Vologda oblast, and Kaluga oblast of Russia. Based on an analysis of the identified nucleotide substitutions and indels in the ITS regions, the M. hypopitys populations were divided into two groups (A and B). Four new ITS haplotypes of M. hypopitys were identified.

Whilst most of the immense species richness of heathers (Calluna, Daboecia and Erica: Ericeae; Ericaceae) is endemic to Africa, particularly the Cape Floristic Region, the oldest lineages are found in the Northern Hemisphere. We present phylogenetic hypotheses for the major clades of Ericeae represented by multiple accessions of all northern Erica species and placeholder taxa for the large nested African/Madagascan clade. We identified consistent, strongly supported conflict between gene trees inferred from ITS and chloroplast DNA sequences with regard to the position of Erica lusitanica. We used coalescent simulations to test whether this conflict could be explained by coalescent stochasticity, as opposed to reticulation (e.g. hybridisation), given estimates of clade ages, generation time and effective population sizes (Ne). A standard approach, comparing overall differences between real and simulated trees, could not clearly reject coalescence. However, additional simulations showed that at the (higher) Ne necessary to explain conflict in E. lusitanica, further topological conflict would also be expected. Ancient hybridisation between ancestors of northern species is therefore a plausible scenario to explain the origin of E. lusitanica, and its morphological similarities to E. arborea. Assuming either process influences the results of species tree and further evolutionary inference. The coalescence scenario is equivocal with regard the standing hypothesis of stepping stone dispersal of Erica from Europe into Africa; whereas reticulate evolution in E. lusitanica would imply that the colonisation of Tropical East Africa by E. arborea instead occurred independently of dispersals within the rest of the African/Madagascan clade., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mountains are often more species-rich than lowlands. This could be the result of migration from lowlands to mountains, of a greater survival rate in mountains, or of a higher diversification rate in mountains. We investigated this question in the globally distributed family Ericaceae, which includes c. 4426 species ranging from sea level to > 5000 m. We predict that the interaction of low specific leaf area (SLA) and montane habitats is correlated with increased diversification rates. A molecular phylogeny of Ericaceae based on rbcL and matK sequence data was built and dated with 18 fossil calibrations and divergence time estimates. We identified radiations using bamm and correlates of diversification rate changes using binary-state speciation and extinction (BiSSE) and multiple-state speciation and extinction (MuSSE) analyses. Analyses revealed six largely montane radiations. Lineages in mountains diversified faster than nonmountain lineages (higher speciation rate, but no difference in extinction rate), and lineages with low SLA diversified faster than high-SLA lineages. Further, habitat and trait had a positive interactive effect on diversification. Our results suggest that the species richness in mountains is the result of increased speciation rather than reduced extinction or increased immigration. Increased speciation in Ericaceae was facilitated by low SLA., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

This is the first study to investigate the secondary structure of 5.8S rRNA in M. hypopitys and related Ericaceae species. The identified nucleotide substitutions are localized mostly at the 3' and 5' ends of the gene, in the region of the third hairpin, and do not significantly affect the topology of the secondary structure of the 5.8S rRNA molecule.

Following climate cooling at the end of the Tertiary, arctic-alpine plants attained most of their extant species diversity. Because East Asia was not heavily glaciated, the importance of this region as a location for the long-term persistence of these species and their subsequent endemism during the Pleistocene was proposed in early discussions of phytogeography. However, this hypothesis remains to be fully tested. Here, we address this hypothesis by elucidating the phylogenetic history of Phyllodoce (Ericaceae). A phylogenetic tree based on multiple nuclear loci revealed that Phyllodoce nipponica was not derived from widespread species such as the arctic-alpine Phyllodoce caerulea, but rather represented an independent lineage sister to the clade of widespread relatives. Molecular dating indicated a mid-Pleistocene divergence of P. nipponica. These findings exclude the hypothesis that P. nipponica was derived from an arctic-alpine species that extended its range southwards during recent glacial periods. Instead, our results support the hypothesis that P. nipponica is an ancestral species which persisted in the Japanese archipelago during the mid- and late Pleistocene. Our findings demonstrate support for the early proposal and shed light on the importance of the Japanese archipelago for the evolution and persistence of arctic-alpine species., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Many habitat specialist species are originally composed of small, discontinuous populations because their habitats are naturally fragmented or patchy. They may have suffered the long-term effects of natural patchiness. Mediterranean heathlands, a representative habitat in the Strait of Gibraltar region, are associated with nutrient-poor, acidic sandstone soils. Sandstone soil patches in the African side of the Strait (Tangier) are, in general, smaller and more scattered than in the European side (Algeciras). In this study, we analyze the effect of this sandstone patchiness on the population genetic diversity and structure of two Erica species from these Mediterranean heathlands that differ in their edaphic specificity, E. australis, sandstone specialist, and E. arborea, generalist. Average levels of within-population genetic diversity and gene flow between populations were significantly lower in Tangier (high sandstone patchiness) than in Algeciras (low patchiness) for the sandstone specialist, whereas no differences between both sides of the Strait were detected in the edaphic generalist. Since most endemic species in Mediterranean heathlands of the Strait of Gibraltar are sandstone specialists, these results highlight an increased vulnerability to loss of genetic diversity and local extinction of the heathland endemic flora in the Tangier side of the Strait of Gibraltar.

Completely sequenced plastomes provide a valuable source of information about the duplication, loss, and transfer events of chloroplast genes and phylogenetic data for resolving relationships among major groups of plants. Moreover, they can also be useful for exploiting chloroplast genetic engineering technology. Ericales account for approximately six per cent of eudicot diversity with 11,545 species from which only three complete plastome sequences are currently available. With the aim of increasing the number of ericalean complete plastome sequences, and to open new perspectives in understanding Mediterranean plant adaptations, a genomic study on the basis of the complete chloroplast genome sequencing of Arbutus unedo and an updated phylogenomic analysis of Asteridae was implemented. The chloroplast genome of A. unedo shows extensive rearrangements but a medium size (150,897 nt) in comparison to most of angiosperms. A number of remarkable distinct features characterize the plastome of A. unedo: five-fold dismissing of the SSC region in relation to most angiosperms; complete loss or pseudogenization of a number of essential genes; duplication of the ndhH-D operon and its location within the two IRs; presence of large tandem repeats located near highly re-arranged regions and pseudogenes. All these features outline the primary evolutionary split between Ericaceae and other ericalean families. The newly sequenced plastome of A. unedo with the available asterid sequences allowed the resolution of some uncertainties in previous phylogenies of Asteridae.

The origins and evolutionary history of the New Zealand flora has been the subject of much debate. The recent description of Cyathodophyllum novaezelandieae from early Miocene sediments in New Zealand provides possible evidence for the antiquity of the fleshy fruited epacrids (tribe Styphelieae, Ericaceae) in New Zealand. Yet the extant species in this tribe are thought to be very closely related to or conspecific with Australian taxa, suggesting recent trans-Tasman origins. In order to investigate the origins and evolution of the extant New Zealand Styphelieae we produced molecular phylogenetic trees based on sequences of three plastid regions that include representatives of all the genera of the tribe and eight of the ten New Zealand species. We estimated the range of minimum ages of the New Zealand lineages with Bayesian relaxed-clock analyses using different calibration methods and relative dating. We found strong support for each of the eight extant species of New Zealand Styphelieae being a distinct lineage that is nested within an Australian clade. In all except one case the sister is from Tasmania and/or the east coast of mainland Australia; for Acrothamnus colensoi the sister is in New Guinea. Estimated dates indicate that all of the New Zealand lineages diverged from their non-New Zealand sisters within the last 7 Ma. Time discontinuity between the fossil C.novae-zelandiae (20-23 Ma) and the origins of the extant New Zealand lineages (none older than 5 Ma) indicates that the fossil and extant Styphelieae in New Zealand are not related. The relative dating analysis showed that to accept this relationship, it would be necessary to accept that the Styphelieae arose in the early-mid Mesozoic (210-120 Ma), which is starkly at odds with multiple lines of evidence on the age of Ericales and indeed the angiosperms. Therefore, our results do not support the hypothesis that Styphelieae have been continuously present in New Zealand since the early Miocene. Instead they suggest a historical biogeographical scenario in which the lineage to which C. novae-zelandiae belongs went extinct in New Zealand, and the extant New Zealand Styphelieae are derived from Australian lineages that recolonised (presumably by long distance dispersal) no earlier than the late Miocene to Pliocene., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Over 60 Pieris taxa (Ericaceae) were measured for their susceptibility to the Andromeda lace bug, Stephanitis takeyai Drake and Maa, and the azalea lace bug, Stephanitis pyrioides (Scott) (Hemiptera: Tingidae) based on leaf damage, adult survival on leaves, and emergence of nymphs in no-choice petri dish assays. Pieris phillyreifolia (Hook.) DC. and P. japonica (Thunb.) D.Don ex G.Don 'Variegata' were consistently resistant to both species of lace bugs, whereas P. japonica 'Cavatine' was consistently susceptible to both. Pieris japonica 'Temple Bells' was highly susceptible to S. takeyai, but resistant to S. pyrioides. Nymph emergence was noted only with S. takeyai, on 46 Pieris taxa, whereas S. pyrioides nymphs were not observed on any of the Pieris taxa. Choice assays (with 10 Pieris taxa) and whole plant assays (with five Pieris taxa) using S. takeyai alone also were conducted, confirming the resistance of P. phillyreifolia and susceptibility of P. japonica Temple Bells to lace bug feeding.

Like other myco-heterotrophic plants, Pterospora andromedea (pinedrops) is dependent upon its specific fungal symbionts for survival. The rarity of pinedrops fungal symbiont was investigated in the eastern United States where pinedrops are rare. Wild populations of eastern pinedrops were sampled, and the plant haplotypes and fungal symbionts were characterized with molecular techniques; these data were compared to those from the West with phylogenetic analyses. The frequency of the fungal symbiont in eastern white pine forests was assessed using a laboratory soil bioassay and in situ pinedrops seed baiting. Only one plant haplotype and fungal symbiont was detected. The plant haplotype was not unique to the East. The fungal symbiont appears to be a new species within the genus Rhizopogon, closely related to the western symbionts. This fungal species was not frequent in soils with or without pinedrops, but was less frequent in the latter and in comparison to the fungal symbionts in western forests. Seed baiting resulted in few germinants, suggesting that mycelial networks produced by the eastern fungal symbiont were rare. Results suggest that eastern pinedrops rarity is influenced by the distribution and rarity of its fungal symbiont.

Premise of the Study: Mixotrophy is a strategy whereby plants acquire carbon both through photosynthesis and heterotrophic exploitation of mycorrhizal fungi. In Euro-American Pyroleae species studied hitherto, heterotrophy levels vary according to species, sites of study, and possibly light conditions. We investigated mycorrhizal association and mixotrophy in the Asiatic forest species Pyrola japonica, and their plasticity under different light conditions., Methods: Pyrola japonica was sampled bimonthly in sunny and shaded conditions from a deciduous broadleaf forest. We microscopically assessed the rate of fungal colonization and sequenced the ITS to identify the mycorrhizal fungi. We measured (13)C and (15)N isotopic abundances in P. japonica as compared with neighboring autotrophic and mycoheterotrophic plants, to evaluate P. japonica's heterotrophy level., Key Results: Pyrola japonica formed arbutoid mycorrhizas devoid of fungal mantles, with intracellular hyphal coils and a Hartig net. It tended to be more colonized by mycorrhizal fungi in spring and summer. Most associated fungi belonged to ectomycorrhizal taxa, and 84% of identified fungi were Russula spp. Rate of mycorrhizal colonization and Russula frequency tended to be higher in shaded conditions. Both δ(13)C and δ(15)N values of P. japonica were significantly higher in autotrophic plants, showing that about half of the carbon on average was received from mycorrhizal fungi. Both isotopic values negatively correlated with light availability, suggesting higher heterotrophy levels in shaded conditions., Conclusions: The mixotrophic P. japonica undergoes changes in mycorrhizal symbionts and carbon nutrition according to light availability. Our results suggest that during Pyroleae evolution, a tendency to increased heterotrophy emerged in the Pyrola/Orthilia clade.

Unlike parasitic plants, which are linked to their hosts directly through haustoria, mycoheterotrophic (MHT) plants derive all or part of their water and nutrients from autothrophs via fungal mycorrhizal intermediaries. Ericaceae, the heather family, are a large and diverse group of plants known to form elaborate symbiotic relationships with mycorrhizal fungi. Using PHYA sequence data, we first investigated relationships among mycoheterotrophic Ericaceae and their close autotrophic relatives. Phylogenetic results suggest a minimum of two independent origins of MHT within this family. Additionally, a comparative investigation of plastid genomes (plastomes) grounded within this phylogenetic framework was conducted using a slot-blot Southern hybridization approach. This survey encompassed numerous lineages of Ericaceae with different life histories and trophic levels, including multiple representatives from mixotrophic Pyroleae and fully heterotrophic Monotropeae and Pterosporeae. Fifty-four probes derived from all categories of protein coding genes typically found within the plastomes of flowering plants were used. Our results indicate that the holo-mycoheterotrophic Ericaceae exhibit extensive loss of genes relating to photosynthetic function and expression of the plastome but retain genes with possible functions outside photosynthesis. Mixotrophic taxa tend to retain most genes relating to photosynthetic functions but are varied regarding the plastid ndh gene content. This investigation extends previous inferences that the loss of the NDH complex occurs prior to becoming holo-heterotrophic and it shows that the pattern of gene losses among mycoheterotrophic Ericaceae is similar to that of haustorial parasites. Additionally, we identify the most desirable candidate species for entire plastome sequencing.

Premise of the Study: Polyploidy is widely recognized as an important process in the evolution of plants, but less attention has been paid to the study of intraspecific polyploidy, including its prevalence, formation, taxonomic implications, and effect on genetic diversity, structure, and gene flow within and among individuals and populations. Here we studied intraspecific ploidy level variation in the Phlox amabilis-P. woodhousei complex to determine the amount and distribution of cytotypic and genetic variation present and measure the extent of gene flow among species, cytotypes, and populations., Methods: Flow cytometry and microsatellite analyses were used to ascertain cytotypic variation, genetic diversity, and population structure within and among eight populations of P. amabilis and 10 populations of P. woodhousei from Arizona and New Mexico., Key Results: Our analyses support the recognition of P. amabilis and P. woodhousei as two distinct species. Both species exhibit cytotypic variation with geographically structured diploid, tetraploid, and hexaploid populations, and genetic analyses suggest a combination of auto- and allopolyploidy in their formation. Diploid, tetraploid, and most hexaploid populations within species share much of their genetic variation, while some hexaploid populations are genetically distinct. All populations maintain moderately high genetic diversity and connectivity, and genetic structure is strongly influenced by geography., Conclusions: This study highlights the potential for complicated patterns of genetic variation relative to cytotypic variation and provides evidence for the role of cytotypic variation and geographic isolation in shaping diversity, differentiation, and potentially speciation in the P. amabilis-P. woodhousei complex.

The perennial evergreen herb, Chimaphila japonica is found exclusively in East Asian temperate coniferous or sometimes in deciduous forests. By using the Fast Isolation by Amplified Fragment Length Polymorphism (AFLP) of Sequences Containing repeats (FIASCO) protocol, 20 microsatellite primer sets were identified in two wild populations. Of these primers, 16 displayed polymorphisms and 4 were monomorphic. The number of alleles per locus ranged from one to six among populations, values for expected and observed heterozygosities ranged from 0.000 to 0.848 and from 0.000 to 1.000, respectively. The new SSR markers will be useful in obtaining estimates of population-level genetic diversity and in phylogeographic studies of C. japonica.

For the predominantly southern hemisphere plant group Styphelioideae (Ericaceae) published sequence datasets of five markers are now available for all except one of the 38 recognised genera. However, several markers are highly incomplete therefore missing data is problematic for producing a genus level phylogeny. We explore the relative utility of supertree and supermatrix approaches for addressing this challenge, and examine the effects of missing data on tree topology and resolution. Although the supertree approach returned a more conservative hypothesis, overall, both supermatrix and supertree analyses concurred in the topologies they returned. Using multiple genes and a dataset of variably complete taxa we found improved support for the monophyly and position of the tribes and genus level relationships. However, there was mixed support for the Richeeae tribe appearing one node basal to the Cosmelieae tribe or vice versa. It is probable that this will only be resolved through further sequencing. Our study supports previous findings that the amount of data is more critical than the completeness of the dataset in estimating well-resolved trees. Our results suggest that a "serendipitous" scaffolding approach that includes a mixture of well and poorly sequenced taxa can lead to robust phylogenetic hypotheses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Erica L. is the largest of the 'Cape' clades that together comprise around half of the disproportionately high species richness of the Cape Floristic Region (CFR) of South Africa. Around 840 species of Erica are currently recognised, C.680 of which are found in the CFR, the rest distributed across the rest of Southern Africa, the highlands of Tropical Africa and Madagascar, and Europe. Erica is taxonomically well documented, but very little is known about species-level relationships. We present the first densely sampled phylogenetic analysis of Erica, using nuclear ribosomal DNA sequences (internal transcribed spacers; ITS) of c. 45% of the species from across the full geographic range of the genus, both Calluna and Daboecia (Ericeae; monotypic genera and putative sister groups of Erica), and further Ericoideae outgroups. Our results show both morphological and geographic coherence of some clades, but numerous shifts in floral macro-morphology as represented by variation in individual morphological characters and pollination syndromes. European Ericeae is a paraphyletic grade subtending a monophyletic African/Malagasy Erica. Given the limited resolution of this single gene tree, more data are needed for further conclusions. Clades are identified that will serve as an effective guide for targeted sampling from multiple linkage groups., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Premise of the Study: Our objective of this study was to develop genomic and expressed sequence tag (EST)-derived microsatellites for the endangered species Rhododendron aureum and access their transferability in R. dauricum and R. brachycarpum. •, Methods and Results: Twelve genomic microsatellites were isolated in R. aureum using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol and seven EST-derived microsatellites were characterized by screening the Rhododendron dbEST database of GenBank. The number of alleles per locus ranged from one to eight. The observed and expected heterozygosity varied from 0.00 to 1.00 and from 0.00 to 0.73, respectively. •, Conclusions: A total of 19 microsatellite loci were developed for R. aureum. Sixteen and 10 of these loci were successfully amplified in R. brachycarpum and R. dauricum, respectively. These microsatellite markers will have potential applications in genetic diversity and conservation genetics studies.

Pyroleae (Ericaceae) consist of four genera, all of which are distributed widely in temperate coniferous or sometimes deciduous forests of the Northern Hemisphere. To investigate the phylogenetic relationships among these genera and to explore the evolution of the characteristics of the subfamily, we conducted maximum parsimony and Bayesian analyses with nrDNA ITS and three cpDNA intergenic spacers (atpB-rbcL, trnS-trnG and trnL-trnF). The results from cpDNA and combined cpDNA + ITS data sets strongly support the monophyly of Pyroleae as well as a sister relationship between Pyrola and Moneses-Chimaphila, with Orthilia as the basal lineage. The sister-group relationship between Moneses and Chimaphila is supported by a set of synapomorphies, e.g., single flower, colpate pollen, five bundles in the style, straight fruiting pedicel orientation, complete capsule dehiscence, and the basic chromosome number, x = 13. The Moneses-Chimaphila-Pyrola clade is supported by at least one homologous character of pollen in tetrads. Conflicts associated with the phylogenetic position of Orthilia may imply a hybrid origin for it, and therefore further study is needed.

Background and Aims: Peripheral populations of plant species are often characterized by low levels of genetic diversity as a result of genetic drift, restricted gene flow, inbreeding and asexual reproduction. These effects can be exacerbated where range-edge populations are fragmented. The main aim of the present study was to assess the levels of genetic diversity in remnant populations of Hypopitys monotropa (syn. Monotropa hypopitys; yellow bird's nest) at the edge of the species' European range in Northern Ireland, since these remnant populations are small and highly fragmented., Methods: Every plant found through surveys of 21 extant populations was genotyped for eight microsatellite loci to estimate levels and patterns of genetic diversity and clonality., Key Results: Levels of genetic diversity were relatively high in the populations studied, and the incidence of clonal reproduction was generally low, with a mean of only 14·45 % of clonal individuals. Clones were small and highly spatially structured. Levels of inbreeding, however, were high., Conclusions: The observed low levels of clonality suggest that the majority of genets in the populations of H. monotropa studied are fertile and that reproduction is predominantly sexual. As the species is highly self-compatible, it is likely that the high levels of inbreeding observed in the populations in the present study are the result of self-pollination, particularly given the small numbers of individuals in most of the patches. Given this extent of inbreeding, further genetic monitoring would be advisable to ensure that genetic diversity is maintained.

Background: The aim of the present study was to use a combined phylogeographic and species distribution modelling approach to compare the glacial histories of two plant species with overlapping distributions, Orthilia secunda (one-sided wintergreen) and Monotropa hypopitys (yellow bird's nest). Phylogeographic analysis was carried out to determine the distribution of genetic variation across the range of each species and to test whether both correspond to the "classic" model of high diversity in the south, with decreasing diversity at higher latitudes, or whether the cold-adapted O. secunda might retain more genetic variation in northern populations. In addition, projected species distributions based on a future climate scenario were modelled to assess how changes in the species ranges might impact on total intraspecific diversity in both cases., Results: Palaeodistribution modelling and phylogeographic analysis using multiple genetic markers (chloroplast trnS-trnG region, nuclear ITS and microsatellites for O. secunda; chloroplast rps2, nuclear ITS and microsatellites for M. hypopitys) indicated that both species persisted throughout the Last Glacial Maximum in southern refugia. For both species, the majority of the genetic diversity was concentrated in these southerly populations, whereas those in recolonized areas generally exhibited lower levels of diversity, particularly in M. hypopitys. Species distribution modelling based on projected future climate indicated substantial changes in the ranges of both species, with a loss of southern and central populations, and a potential northward expansion for the temperate M. hypopitys., Conclusions: Both Orthilia secunda and Monotropa hypopitys appear to have persisted through the LGM in Europe in southern refugia. The boreal O. secunda, however, has retained a larger proportion of its genetic diversity in more northerly populations outside these refugial areas than the temperate M. hypopitys. Given that future species distribution modelling suggests northern range shifts and loss of suitable habitat in the southern parts of the species' current distributions, extinction of genetically diverse rear edge populations could have a significant effect in the rangewide intraspecific diversity of both species, but particularly in M. hypopitys.

Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds-namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 μg/μL and the purity, evaluated by the ratio A(260)/A(280), was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR.

Arbutus unedo grows spontaneously around the Mediterranean basin. The species is tolerant to drought and has a strong regeneration capacity following fires making it interesting for Mediterranean forestation programs. Considering the sparse information about the potential of this fruit tree to be propagated in vitro, a project to clone selected trees based on their fruit production was initiated a few years ago. The role of several factors on A. unedo propagation was evaluated. The results showed that 8.9 μm kinetin gave the best results although not significantly different from those obtained with benzyladenine or zeatin. The inclusion of thidiazuron or 1-naphthaleneacetic acid promoted callus growth and had deleterious effects on the multiplication rate. The genotype of the donor plants is also a factor interfering with the multiplication. The results also indicated that the conditions used for multiplication influenced the behavior of shoots during the rooting phase., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Understanding the processes of biological diversification is a central topic in evolutionary biology. The South African Cape fynbos, one of the major plant biodiversity hotspots out of the tropics, has prompted several hypotheses about the causes of generation and maintenance of biodiversity. Fire has been traditionally invoked as a key element to explain high levels of biodiversity in highly speciose fynbos taxa, such as the genus Erica. In this study, we have implemented a microevolutionary approach to elucidate how plant-response to fire may contribute to explain high levels of diversification in Erica. By using microsatellite markers, we investigated the genetic background of seeder (fire-sensitive) and resprouter (fire-resistant) populations of the fynbos species Erica coccinea. We found higher within-population genetic diversity and higher among-population differentiation in seeder populations and interpreted these higher levels of genetic diversification as a consequence of the comparatively shorter generation times and faster population turnover in the seeder form of this species. Considering that genetic divergence among populations may be seen as the initial step to speciation, the parallelism between these results and the pattern of biodiversity at the genus level offers stimulating insights into understanding causes of speciation of the genus Erica in the Cape fynbos., (© 2010 The Author(s). Evolution© 2010 The Society for the Study of Evolution.)

Phytochromes play a key role in allowing plants to monitor their surrounding environment and, conversely, adaptation to local environments has driven the evolutionary history of phytochromes. As a result of natural selection, polymorphisms in phytochrome genes would thus be expected to exhibit locus-specific phylogeographic structure. To evaluate this hypothesis, we conducted a phylogeographic investigation based on four nuclear genes, including two phytochrome genes (PHYB and PHYE) using 155 samples of Arcterica nana from the entire range of the Japanese archipelago. Bayesian clustering revealed geographic differentiation between northern and southern Japan when all four genes were included. However, this geographic differentiation is inconsistent with previously reported genetic structure of genome-wide polymorphisms based on amplified fragment length polymorphisms, as these did not show geographic differentiation throughout the Japanese archipelago. In contrast, the north-south differentiation was not apparent when PHYE was excluded. This indicates that PHYE alone could be responsible for the north-south differentiation (F(CT) = 0.15, P < 0.001). Furthermore, a single nonsynonymous polymorphism (C360T) strongly contributed to geographic differentiation (F(CT) = 0.57, P < 0.001) and its corresponding amino acid replacement (P120L) was significantly under positive selection based on maximum likelihood analysis (P = 0.98). Consequently, the locus-specific geographic differentiation in PHYE could be caused by natural selection, suggesting the involvement of PHYE in local adaptation between populations of A. nana in northern and southern Japan. This finding is consistent with a previous study on Cardamine nipponica, indicating the importance of PHYE for local adaptation in Japanese alpine plants.

Subfamily Ericoideae (Ericaceae) includes 19 genera in five recognized tribes. Relationships involving the deepest nodes have been difficult to resolve, limiting the potential for further cladistic studies within the Ericoideae. The current study analyses six molecular markers using Bayesian, Maximum Likelihood and Maximum Parsimony methods to improve phylogenetic resolution within the Ericoideae. Two large clades were discovered. One clade includes the Phyllodoceae and Bejaria. The sister clade includes the Empetreae+Diplarche, Ericeae, Rhodoreae, and a clade comprised of Bryanthus and Ledothamnus. The current study improves upon the resolution of the phylogeny of the Ericoideae, particularly demonstrating support for the deepest nodes. Based on these results, we propose to retain the Ericeae, expand the Phyllodoceae to include Bejaria, expand the Empetreae to include Diplarche, retain the Rhodoreae (without Diplarche), dismantle the Bejarieae, and construct a new tribe, Bryantheae (Bryanthus and Ledothamnus)., (Copyright 2010 Elsevier Inc. All rights reserved.)