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.

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.

A chloroplast tree for Viburnum (Adoxaceae) and its implications for phylogenetic classification and character evolution.

• Premise of the study: Despite recent progress, significant uncertainties remain concerning relationships among early-branching lineages within Viburnum (Adoxaceae), prohibiting a new classification and hindering studies of character evolution and the increasing use of Viburnum in addressing a wide range of ecological and evolutionary questions. We hoped to resolve these issues by sequencing whole plastid genomes for representative species and combining these with molecular data previously obtained from an expanded taxon sample.• Methods: We performed paired-end Illumina sequencing of plastid genomes of 22 Viburnum species and combined these data with a 10-gene data set to infer phylogenetic relationships for 113 species. We used the results to devise a comprehensive phylogenetic classification and to analyze the evolution of eight morphological characters that vary among early-branching lineages.• Key results: With greatly increased levels of confidence in most of the early branches, we propose a phylogenetic classification of Viburnum, providing formal phylogenetic definitions for 30 clades, including 13 with names recognized under the International Code of Nomenclature for Algae, Fungi, and Plants, eight with previously proposed informal names, and nine newly proposed names for major branches. Our parsimony reconstructions of bud structure, leaf margins, inflorescence form, ruminate endosperm, extrafloral nectaries, glandular trichomes, palisade anatomy, and pollen exine showed varying levels of homoplasy, but collectively provided morphological support for some, though not all, of the major clades.• Conclusions: Our study demonstrates the value of next-generation plastid sequencing, the ease of creating a formal phylogenetic classification, and the utility of such a system in describing patterns of character evolution.

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.

Phylogenetic and experimental tests of interactions among mutualistic plant defense traits in Viburnum (adoxaceae).

Plant traits that mediate mutualistic interactions are widespread, yet few studies have linked their macroevolutionary patterns with the ecological interactions they mediate. Here we merged phylogenetic and experimental approaches to investigate the evolution of two common mutualistic plant traits, extrafloral nectaries (EFNs) and leaf domatia. By using the flowering plant clade Viburnum, we tested whether macroevolutionary patterns support adaptive hypotheses and conducted field surveys and manipulative experiments to examine whether ecological interactions are concordant with evolutionary predictions. Phylogenetic reconstructions suggested that EFN-bearing species are monophyletic, whereas the evolution of domatia correlated with leaf production strategy (deciduous or evergreen) and climate. Domatia were also more common in the EFN clade, suggesting that the two traits may jointly mediate ecological interactions. This result was further investigated in a common-garden survey, where plants with domatia and EFNs on the leaf blade had more mutualistic mites than plants with other trait combinations, and in manipulative field experiments, where the traits additively increased mutualist abundance. Taken together, our results suggest that mutualistic traits in Viburnum are not ecologically independent, as they work in concert to attract and retain mutualists, and their long-term evolution may be influenced by complex interactions among multiple traits, mutualists, and geography.

Determination of the total phenolic and anthocyanin contents and antimicrobial activity of Viburnum opulus fruit juice.

Viburnum opulus is a plant with fruits that are rich in biologically active substances, making it valuable to the food and pharmaceutical industries. Here, we present our study of the total phenolic and anthocyanin contents and antimicrobial activity in the fruit juice of six V. opulus L. accessions. The cultivar 'Krasnaya Grozd' was notable for its exceptionally large amount of total phenolics, 1168 mg/100 g, with anthocyanins comprising 3-5 % of the total phenolic content. The evaluation of the antimicrobial properties confirmed that the juice of V. opulus fruits strongly inhibited the growth of a wide range of human pathogenic bacteria, both Gram-negative (Salmonella typhimurium and S. agona) and Gram-positive (Staphylococcus aureus, Lysteria monocytogenes, and Enterococcus faecalis) organisms. Conversely, the yeasts Debaryomyces hansenii and Torulaspora delbrueckii showed complete resistance to the fruit juice, whereas a low sensitivity was demonstrated by Trichosporon cutaneum, Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, S. cerevisiae 12R, and Candida parapsilosis.