Complete genome analysis of a novel narnavirus in sweet viburnum (Viburnum odoratissimum).

Trees and shrubs provide important ecological services. However, few studies have surveyed the virome in trees and shrubs. In this study, we discovered a new positive-sense RNA virus originating from Viburnum odoratissimum, which we named "Vo narna-like virus". The complete genome of Vo narna-like virus is 3,451 nt in length with an open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRP) protein. Phylogenetic analysis placed this virus within the betanarnavirus clade, sharing 53.63% amino acid sequence identity with its closest relative, Qingdao RNA virus 2. The complete sequence of the virus was confirmed by rapid amplification of cDNA ends (RACE) and Sanger sequencing. Small interfering RNA (siRNA) analysis indicated that this virus interacts with the RNA interference (RNAi) pathway of V. odoratissimum. This is the first report of a narnavirus in V. odoratissimum.

Assembly and comparative analysis of the complete mitochondrial genome of Viburnum chinshanense.

BACKGROUND: Viburnum chinshanense is an endemic species found exclusively in the North-Central and South-Central regions of China. This species is a lush garden ornamental tree and is extensively utilized for vegetation restoration in rocky desertification areas. RESULTS: In this study, we obtained 13.96 Gb of Oxford Nanopore data for the whole genome, and subsequently, by combining Illumina short-reads, we successfully assembled the complete mitochondrial genome (mitogenome) of the V. chinshanense using a hybrid assembly strategy. The assembled genome can be described as a circular genome. The total length of the V. chinshanense mitogenome measures 643,971 bp, with a GC content of 46.18%. Our annotation efforts have revealed a total of 39 protein-coding genes (PCGs), 28 tRNA genes, and 3 rRNA genes within the V. chinshanense mitogenome. The analysis of repeated elements has identified 212 SSRs, 19 long tandem repeat elements, and 325 pairs of dispersed repeats in the V. chinshanense mitogenome. Additionally, we have investigated mitochondrial plastid DNAs (MTPTs) and identified 21 MTPTs within the mitogenome and plastidial genome. These MTPTs collectively span a length of 9,902 bp, accounting for 1.54% of the mitogenome. Moreover, employing Deepred-mt, we have confidently predicted 623 C to U RNA editing sites across the 39 protein-coding genes. Furthermore, extensive genomic rearrangements have been observed between V. chinshanense and the mitogenomes of related species. Interestingly, we have also identified a bacterial-derived tRNA gene (trnC-GCA) in the V. chinshanense mitogenome. Lastly, we have inferred the phylogenetic relationships of V. chinshanense with other angiosperms based on mitochondrial PCGs. CONCLUSIONS: This study marks the first report of a mitogenome from the Viburnum genus, offering a valuable genomic resource for exploring the evolution of mitogenomes within the Dipsacales order.

A set of SSR markers to characterize genetic diversity in all Viburnum species.

About 160 species are classified within the Viburnum genus and many of these are cultivated for horticultural purposes. The vast dispersal of Viburnum makes the genus a useful model for studying evolutionary history and inferring how species expanded into their current distributions. Simple sequence repeat (SSR) markers were previously developed for five Viburnum species that were classified within the four major clades (Laminotinus, Crenotinus, Valvatotinus, and Porphyrotinus). The ability of some of these markers to cross-amplify in Viburnum species has been scantly evaluated, but there has not been any genus-wide assessment for the markers. We evaluated a collection of 49 SSR markers for the ability to cross-amplify in 224 samples, including 46 Viburnum species, representing all 16 subclades, and five additional species in the Viburnaceae and Caprifoliaceae. A subset of 14 potentially comprehensive markers for Viburnum species was identified and evaluated for the ability to detect polymorphisms in species outside of their respective clades. The 49 markers had overall amplification success in 52% of the samples, including a 60% success rate within the Viburnum genus and 14% in other genera. The comprehensive marker set amplified alleles in 74% of all samples tested, including 85% of Viburnum samples and 19% of outgroup samples. To the best of our knowledge, this is the first comprehensive set of markers able to characterize species across an entire genus. This set of markers can be used to assess the genetic diversity and population structure of most Viburnum species and closely allied species.

Within-individual leaf allometry and the evolution of leaf morphology: A multilevel analysis of leaf allometry in temperate Viburnum (Adoxaceae) species.

A critical issue in evolutionary biology is understanding the relationship between macroevolutionary patterns of diversity and the origin of variation at the organismal level. Among-individual allometry, the relationship between the size and shape of a structure among organisms at a fixed developmental stage, is often similar to evolutionary allometry, the relationship between the size and shape of a structure among populations or species, and the genetic and developmental process that underlie allometric relationships at both levels are thought to influence evolutionary diversification. Metameric organisms present an additional level of allometry: the relationship between the size and shape of structures within individuals. We propose that within-individual allometry is also related to evolutionary diversification among metameric organisms. We explore this idea in temperate deciduous Viburnum (Adoxaceae) species that bear two types of leaves, that is, preformed and neoformed leaves, with contrasting patterns of development. Examination of within-individual, among-individual, among-population, and among-species allometry of leaf shape in both leaf types showed that the slopes of all allometric relationships were significantly different from isometry, and their sign was consistent across allometric hierarchies. Although the allometric slope of preformed leaves was constant across allometry levels, the allometric slope of neoformed leaves became increasingly steeper. We suggest that allometric variation underlying evolutionary diversification in metameric organisms may manifest among individuals and also among their repeated structures. Moreover, structures with contrasting patterns of development within metameric organisms can experience different degrees of developmental constraint, and this can in turn affect morphological diversification.

Viburnum tinus Fruits Use Lipids to Produce Metallic Blue Structural Color.

Viburnum tinus is an evergreen shrub that is native to the Mediterranean region but cultivated widely in Europe and around the world. It produces ripe metallic blue fruits throughout winter [1]. Despite its limited fleshy pulp [2], its high lipid content [3] makes it a valuable resource to the small birds [4] that act as its seed-dispersers [5]. Here, we find that the metallic blue appearance of the fruits is produced by globular lipid inclusions arranged in a disordered multilayer structure. This structure is embedded in the cell walls of the epicarp and underlaid with a dark layer of anthocyanin pigments. The presence of such large, organized lipid aggregates in plant cell walls represents a new mechanism for structural coloration and may serve as an honest signal of nutritional content.

Fruit syndromes in Viburnum: correlated evolution of color, nutritional content, and morphology in bird-dispersed fleshy fruits.

PREMISE: A key question in plant dispersal via animal vectors is where and why fruit colors vary between species and how color relates to other fruit traits. To better understand the factors shaping the evolution of fruit color diversity, we tested for the existence of syndromes of traits (color, morphology, and nutrition) in the fruits of Viburnum. We placed these results in a larger phylogenetic context and reconstructed ancestral states to assess how Viburnum fruit traits have evolved across the clade. RESULTS: We find that blue Viburnum fruits are not very juicy, and have high lipid content and large, round endocarps surrounded by a small quantity of pulp. Red fruits display the opposite suite of traits: they are very juicy with low lipid content and smaller, flatter endocarps. The ancestral Viburnum fruit may have gone through a sequence of color changes before maturation (green to yellow to red to black), though our reconstructions are equivocal. In one major clade of Viburnum (Nectarotinus), fruits mature synchronously with reduced intermediate color stages. Most transitions between fruit colors occurred in this synchronously fruiting clade. CONCLUSIONS: It is widely accepted that fruit trait diversity has primarily been driven by the differing perceptual abilities of bird versus mammal frugivores. Yet within a clade of largely bird-dispersed fruits, we find clear correlations between color, morphology, and nutrition. These correlations are likely driven by a shift from sequential to synchronous development, followed by diversification in color, nutrition, and morphology. A deeper understanding of fruit evolution within clades will elucidate the degree to which such syndromes structure extant fruit diversity.

Phylogeography of a widespread eastern North American shrub, Viburnum lantanoides.

PREMISE OF THE STUDY: There have been relatively few phylogeographic studies of eastern North American plants, especially of animal-dispersed shrubby species, and this leaves a significant gap in our understanding of how such species were affected by glacial events. Here, we analyzed the phylogeography of the widespread understory shrub Viburnum lantanoides. METHODS: We generated RADseq data and paleoclimatic species distribution models (SDMs) to identify the locations of refugia where V. lantanoides may have survived the Last Glacial Maximum (LGM) and how its range expanded as glaciers receded. KEY RESULTS: Genetic diversity falls off with increasing latitude and longitude, indicating that range expansion likely occurred via serial founder events from southern source populations. Samples from the southern Appalachians form a grade, while those from the north form a clade, suggesting that a single genetic lineage recolonized the north. SDMs indicate that V. lantanoides probably survived the LGM in refugia on the mid-Atlantic Coastal Plain and/or the interior Gulf Coastal Plain. CONCLUSIONS: Our analyses indicate that V. lantanoides survived the LGM in refugia south of the glacier but north of the extensive refugium along the Gulf Coast. Following the LGM, a single population expanded northward along the Appalachian Mountains and eventually into eastern Canada. The patterns observed here suggest that range expansion occurred in a stepwise manner, similar to postglacial dynamics observed in a number of European plant species.

Sterile marginal flowers increase visitation and fruit set in the hobblebush (Viburnum lantanoides, Adoxaceae) at multiple spatial scales.

Background and Aims: Enlarged sterile flowers on the periphery of inflorescences increase the attractiveness of floral displays, and previous studies have generally demonstrated that these have positive effects on insect visitation and/or reproductive success. However, experiments have not specifically been designed to examine the benefits of sterile flowers under conditions that reflect the early stages in their evolution, i.e. when plants that produce sterile flowers are at low frequency. Methods: Over three years, three experiments were performed in natural populations of Viburnum lantanoides, which produces sterile marginal flowers (SMFs). The first experiment established that fruit production in V. lantanoides increases with the receipt of outcross pollen. The second tested the role of SMFs under extant conditions, comparing fruit production in two populations composed entirely of intact plants or entirely of plants with the SMFs removed. The third was designed to mimic the presumed context in which SMFs first evolved; here, SMFs were removed from all but a few plants in a population, and rates of insect visitation and fruit set were compared between plants with intact and denuded SMFs. Key Results: In comparing whole populations, the presence of SMFs nearly doubled fruit set. Under simulated 'ancestral' conditions within a population, plants with intact SMFs received double the insect visits and produced significantly more fruits than denuded plants. There was no significant effect of the number of inflorescences or fertile flowers on insect visitation or fruit set, indicating that the presence of SMFs accounted for these differences. Conclusions: The presence of SMFs significantly increased pollinator attraction and female reproductive success both in contemporary and simulated ancestral contexts, indicating that stabilizing selection is responsible for their maintenance, and directional selection likely drove their evolution when they first appeared. This study demonstrates a novel approach to incorporating historically relevant scenarios into experimental studies of floral evolution.

Restriction-Site-Associated DNA Sequencing Reveals a Cryptic Viburnum Species on the North American Coastal Plain.

Species are the starting point for most studies of ecology and evolution, but the proper circumscription of species can be extremely difficult in morphologically variable lineages, and there are still few convincing examples of molecularly informed species delimitation in plants. Here, we focus on the Viburnum nudum complex, a highly variable clade that is widely distributed in eastern North America. Taxonomic treatments have mostly divided this complex into northern (V. nudum var. cassinoides) and southern (V. nudum var. nudum) entities, but additional names have been proposed. We used multiple lines of evidence, including RADseq, morphological, and geographic data, to test how many independently evolving lineages exist within the V. nudum complex. Genetic clustering and phylogenetic methods revealed three distinct groups-one lineage that is highly divergent, and two others that are recently diverged and morphologically similar. A combination of evidence that includes reciprocal monophyly, lack of introgression, and discrete rather than continuous patterns of variation supports the recognition of all three lineages as separate species. These results identify a surprising case of cryptic diversity in which two broadly sympatric species have consistently been lumped in taxonomic treatments. The clarity of our findings is directly related to the dense sampling and high-quality genetic data in this study. We argue that there is a critical need for carefully sampled and integrative species delimitation studies to clarify species boundaries even in well-known plant lineages. Studies following the model that we have developed here are likely to identify many more cryptic lineages and will fundamentally improve our understanding of plant speciation and patterns of species richness.

Leaf Form Evolution in Viburnum Parallels Variation within Individual Plants.

Few studies have critically evaluated how morphological variation within individual organisms corresponds to variation within and among species. Subindividual variation in plants facilitates such studies because their indeterminate modular growth generates multiple serially homologous structures along growing axes. Focusing on leaf form, we evaluate how subindividual trait variation relates to leaf evolution across Viburnum, a clade of woody angiosperms. In Viburnum we infer multiple independent origins of wide/lobed leaves with toothed margins from ancestors with elliptical, smooth-margined leaves. We document leaf variation along the branches of individual plants of 28 species and among populations across the wide range of Viburnum dentatum. We conclude that when novel leaf forms evolved in Viburnum, they were intercalated at the beginning of the seasonal leaf sequence, which then generated a repeated spectrum of leaf forms along each branch (seasonal heteroblasty). We hypothesize that the existence of such a spectrum then facilitated additional evolutionary shifts, including reversions to more ancestral forms. We argue that the recurrent production of alternative phenotypes provides opportunities to canalize the production of particular forms and that this phenomenon has played an important role in generating macroscale patterns.

miRNAs involved in the development and differentiation of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri.

BACKGROUND: Sterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear. RESULTS: Here, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of Viburnum macrocephalum f. keteleeri. We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in V. macrocephalum f. keteleeri flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while SPL genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be SPL genes, we propose that miR156 may be involved in the regulation of stamen development in V. macrocephalum f. keteleeri. CONCLUSIONS: We identified miRNAs differentially expressed between fertile and sterile flowers in V. macrocephalum f. keteleeri and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.

Temperate radiations and dying embers of a tropical past: the diversification of Viburnum.

We used a near-complete phylogeny for the angiosperm clade Viburnum to assess lineage diversification rates, and to examine possible morphological and ecological factors driving radiations. Maximum-likelihood and Bayesian approaches identified shifts in diversification rate and possible links to character evolution. We inferred the ancestral environment for Viburnum and changes in diversification dynamics associated with subsequent biome shifts. Viburnum probably diversified in tropical forests of Southeast Asia in the Eocene, with three subsequent radiations in temperate clades during the Miocene. Four traits (purple fruits, extrafloral nectaries, bud scales and toothed leaves) were statistically associated with higher rates of diversification. However, we argue that these traits are unlikely to be driving diversification directly. Instead, two radiations were associated with the occupation of mountainous regions and a third with repeated shifts between colder and warmer temperate forests. Early-branching depauperate lineages imply that the rare lowland tropical species are 'dying embers' of once more diverse lineages; net diversification rates in Viburnum likely decreased in these tropical environments after the Oligocene. We suggest that 'taxon pulse' dynamics might characterize other temperate plant lineages.

Genetic variation in host plants influences the mate preferences of a plant-feeding insect.

Many species spend their lives in close association with other organisms, and the environments provided by those organisms can play an important role as causes of variation in phenotypes. When this is the case, the genotypes of the individuals constituting the environment may influence the phenotypes of individuals living in that environment. When these effects are between heterospecifics, interspecific indirect genetic effects (IIGEs) occur. Several studies have detected IIGEs, but whether IIGEs contribute to variation in sexually selected traits remains virtually unexplored. We assessed how mate preferences in a plant-feeding insect are influenced by the genotype of their host plant. We established clone lines of a sample of host plant genotypes constituting the background biotic environment for a random sample of insects that we reared on them. We found that the insects' mate preferences varied according to the clone line on which they developed. These results demonstrate that genetic variation in host plants has cross-trophic consequences on a trait that has strong effects on fitness and interpopulation dynamics such as diversification in communication systems. We discuss how IIGEs on mate preferences may influence the way in which selection acts, including the maintenance of variation and the promotion of evolutionary divergence.

Trees to treehoppers: genetic variation in host plants contributes to variation in the mating signals of a plant-feeding insect.

Community genetics research has demonstrated 'bottom-up' effects of genetic variation within a plant species in shaping the larger community with which it interacts, such as compositions of arthropod faunas. We demonstrate that such cross-trophic interactions also influence sexually selected traits. We used a member of the Enchenopa binotata species complex of treehoppers (Hemiptera: Membracidae) to ask whether male mating signals are influenced by host plant genetic variation. We reared a random sample of the treehoppers on potted replicates of a sample of host plant clone lines. We found that treehopper male signals varied according to the clone line on which they developed, showing that genetic variation in host plants affects male treehoppers' behavioural phenotypes. This is the first demonstration of cross-trophic indirect genetic effects on a sexually selected trait. We discuss how such effects may play an important role in the maintenance of variation and within-population phenotypic differentiation, thereby promoting evolutionary divergence.

Antioxidant properties and polyphenolic compositions of fruits from different European cranberrybush (Viburnum opulus L.) genotypes.

Antioxidant properties of fruit juices of six Viburnum opulus genotypes were evaluated by DPPH, ABTS(+) radical scavenging capacity (RSC), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and Folin-Ciocalteu total phenolic content (TPC) assays. TPC varied in the range of 5.4-10.6 mg gallic acid equivalents/g, RSC (ABTS(+)), FRAP and ORAC values were 31.9-109.8, 32.3-61.8 and 141.6-260.4 µmol trolox equivalents/g, respectively. V. opulus var. sargentii fruit juice was a remarkably stronger antioxidant than the other five V. opulus genotypes. The content of chlorogenic acid (the main phenolic compound in berry juices) depending on plant cultivar varied in the range of 0.54-6.93 mg/ml. The RSC of individual constituents was measured by the on-line HPLC-UV-DPPH method: chlorogenic acid was the dominant radical scavenger in V. opulus P3 (74%), while epicatechin and catechin (the main antioxidants in V. opulus var. sargentii) contributed to 40% and 23% of the total RSC for the sargentii genotype. Nine constituents were identified in V. opulus juice by using ultra high performance liquid chromatography coupled to quadruple and time-of-flight mass spectrometers (UPLC-QTOF-MS). In general, the study demonstrated that V. opulus var. sargentii followed by V. opulus P3 and V. opulus var. americanum possessed the highest antioxidant capacity. The obtained results may assist in selecting the most valuable V. opulus genotypes for the production of fruits possessing strong antioxidant capacity and containing beneficial phenolic constituents.

Evolution of leaf form correlates with tropical-temperate transitions in Viburnum (Adoxaceae).

Strong latitudinal patterns in leaf form are well documented in floristic comparisons and palaeobotanical studies. However, there is little agreement about their functional significance; in fact, it is still unknown to what degree these patterns were generated by repeated evolutionary adaptation. We analysed leaf form in the woody angiosperm clade Viburnum (Adoxaceae) and document evolutionarily correlated shifts in leafing habit, leaf margin morphology, leaf shape and climate. Multiple independent shifts between tropical and temperate forest habitats have repeatedly been accompanied by a change between evergreen, elliptical leaves with entire margins and deciduous, more rounded leaves with toothed or lobed margins. These consistent shifts in Viburnum support repeated evolutionary adaptation as a major determinant of the global correlation between leaf form and mean annual temperature. Our results provide a new theoretical grounding for the inference of past climates using fossil leaf assemblages.

Barcoding success as a function of phylogenetic relatedness in Viburnum, a clade of woody angiosperms.

BACKGROUND: The chloroplast genes matK and rbcL have been proposed as a "core" DNA barcode for identifying plant species. Published estimates of successful species identification using these loci (70-80%) may be inflated because they may have involved comparisons among distantly related species within target genera. To assess the ability of the proposed two-locus barcode to discriminate closely related species, we carried out a hierarchically structured set of comparisons within Viburnum, a clade of woody angiosperms containing ca. 170 species (some 70 of which are currently used in horticulture). For 112 Viburnum species, we evaluated rbcL + matK, as well as the chloroplast regions rpl32-trnL, trnH-psbA, trnK, and the nuclear ribosomal internal transcribed spacer region (nrITS). RESULTS: At most, rbcL + matK could discriminate 53% of all Viburnum species, with only 18% of the comparisons having genetic distances >1%. When comparisons were progressively restricted to species within major Viburnum subclades, there was a significant decrease in both the discriminatory power and the genetic distances. trnH-psbA and nrITS show much higher levels of variation and potential discriminatory power, and their use in plant barcoding should be reconsidered. As barcoding has often been used to discriminate species within local areas, we also compared Viburnum species within two regions, Japan and Mexico and Central America. Greater success in discriminating among the Japanese species reflects the deeper evolutionary history of Viburnum in that area, as compared to the recent radiation of a single clade into the mountains of Latin America. CONCLUSIONS: We found very low levels of discrimination among closely related species of Viburnum, and low levels of variation in the proposed barcoding loci may limit success within other clades of long-lived woody plants. Inclusion of the supplementary barcodes trnH-psbA and nrITS increased discrimination rates but were often more effective alone rather than in combination with rbcL + matK. We surmise that the efficacy of barcoding in plants has often been overestimated because of the lack of comparisons among closely related species. Phylogenetic information must be incorporated to properly evaluate relatedness in assessing the utility of barcoding loci.

Activity, polypeptide and gene identification of thylakoid Ndh complex in trees: potential physiological relevance of fluorescence assays.

Three evergreen (Laurus nobilis, Viburnum tinus and Thuja plicata) and two autumnal abscission deciduous trees (Cydonia oblonga and Prunus domestica) have been investigated for the presence (zymogram and immunodetection) and functionality (post-illumination chlorophyll fluorescence) of the thylakoid Ndh complex. The presence of encoding ndh genes has also been investigated in T. plicata. Western assays allowed tentative identification of zymogram NADH dehydrogenase bands corresponding to the Ndh complex after native electrophoresis of solubilized fractions from L. nobilis, V. tinus, C. oblonga and P. domestica leaves, but not in those of T. plicata. However, Ndh subunits were detected after SDS-PAGE of thylakoid solubilized proteins of T. plicata. The leaves of the five plants showed the post-illumination chlorophyll fluorescence increase dependent on the presence of active Ndh complex. The fluorescence increase was higher in autumn in deciduous, but not in evergreen trees, which suggests that the thylakoid Ndh complex could be involved in autumnal leaf senescence. Two ndhB genes were sequenced from T. plicata that differ at the 350 bp 3' end sequence. Comparison with the mRNA revealed that ndhB genes have a 707-bp type II intron between exons 1 (723 bp) and 2 (729 bp) and that the UCA 259th codon is edited to UUA in mRNA. Phylogenetically, the ndhB genes of T. plicata group close to those of Metasequoia, Cryptomeria, Taxodium, Juniperus and Widdringtonia in the cupresaceae branch and are 5' end shortened by 18 codons with respect to that of angiosperms.

Genetic analysis of an interspecific cross in ornamental Viburnum (Viburnum).

A genetic analysis was performed on a population derived from crosses between Viburnum lantana and Viburnum carlesii. Linkage maps were developed for each species using AFLP, random amplified polymorphic DNA (RAPD), and sequence-tagged site markers and a half-sib approach that took advantage of both the polymorphism between the species and the heterozygosity within each parent. The map for V. lantana consisted of 153 DNA markers and spanned approximately 750 cM, whereas that for V. carlesii contained 133 markers and covered 700 cM. These maps were used to determine the location of several major genes influencing leaf spot resistance, Verticillium wilt resistance, bud color, and flower scent. Both species contained moderate levels of heterozygosity. Flow cytometric analysis revealed that the genome of V. lantana was 40% larger than that of V. carlesii, and this difference was paralleled by a proportionally greater number of intercross markers (markers segregating 3:1) from V. lantana than from V. carlesii. In addition, V. lantana (n = 9) displayed a 10th linkage group for which no homolog in V. carlesii (n = 9) could be found and which contained only markers present in the former species and absent in the latter. These results suggest that Viburnum could be an interesting genetic model for Caprifoliaceae sensu lato.

Computational and experimental analyses of furcatin hydrolase for substrate specificity studies of disaccharide-specific glycosidases.

Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific beta-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyse the beta-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyse monosaccharide glycosides. We analysed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.2.1.161) derived from Viburnum furcatum. Amino acid sequence comparisons and model structure building revealed two residues, Ala419 and Ser504 of FH, as candidates determining the substrate specificity. These residues were specifically conserved in the diglycosidases. The model structure suggested that Ala419 is involved in the aglycone recognition, whereas Ser504 recognizes the external saccharide of the glycone. Mutations at these sites drastically decreased the diglycosidase activity. The mechanism by which the diglycosidases acquired their substrate specificity is discussed, based on these observations.