Geometric morphometrics reveals shifts in flower shape symmetry and size following gene knockdown of CYCLOIDEA and ANTHOCYANIDIN SYNTHASE.

BACKGROUND: While floral symmetry has traditionally been assessed qualitatively, recent advances in geometric morphometrics have opened up new avenues to specifically quantify flower shape and size using robust multivariate statistical methods. In this study, we examine, for the first time, the ability of geometric morphometrics to detect morphological differences in floral dorsoventral asymmetry following virus-induced gene silencing (VIGS). Using Fedia graciliflora Fisch. & Meyer (Valerianaceae) as a model, corolla shape of untreated flowers was compared using canonical variate analysis to knockdown phenotypes of CYCLOIDEA2A (FgCYC2A), ANTHOCYANIDIN SYNTHASE (FgANS), and empty vector controls. RESULTS: Untreated flowers and all VIGS treatments were morphologically distinct from each other, suggesting that VIGS may cause subtle shifts in floral shape. Knockdowns of FgCYC2A were the most dramatic, affecting the position of dorsal petals in relation to lateral petals, thereby resulting in more actinomorphic-like flowers. Additionally, FgANS knockdowns developed larger flowers with wider corolla tube openings. CONCLUSIONS: These results provide a method to quantify the role that specific genes play in the developmental pathway affecting the dorsoventral axis of symmetry in zygomorphic flowers. Additionally, they suggest that ANS may have an unintended effect on floral size and shape.

Rapid isolation and cross-amplification of microsatellite markers in Plectritis congesta (Valerianaceae) with 454 sequencing.

PREMISE OF THE STUDY: Microsatellite markers were isolated and characterized in Plectritis congesta for studying the evolution of this highly variable species. METHODS AND RESULTS: We used 454 sequencing of DNA enriched for microsatellite repeats to develop microsatellite markers. This produced 262079 reads with an average length of 324 bp, representing approximately 800 microsatellite regions from which 48 primers were tested. Eleven markers reliably amplified without optimization. These primer pairs showed a high degree of heterozygosity and allelic diversity. Unexpectedly, half of the markers contained multiple peaks, with up to four alleles per individual, which suggests that either polyploidy or isolated gene duplication has occurred within this clade. These primers successfully cross-amplified in P. macrocera, indicating the utility of these markers for the genus. CONCLUSIONS: With variation in mating system and habitat, a mix of duplicated and nonduplicated markers, and high genetic variance, Plectritis is an ideal candidate model genus for studying the ecological and evolutionary consequences of gene duplication.

Correlates of diversification in the plant clade Dipsacales: geographic movement and evolutionary innovations.

We explore patterns of diversification in the plant clades Adoxaceae and Valerianaceae (within Dipsacales), evaluating correlations between biogeographic change (i.e., movements into new areas), morphological change (e.g., the origin of putative key innovations associated with vegetative and reproductive characters), and shifts in rates of diversification. Our findings indicate that rates of diversification in these plants tend to be less tightly correlated with the evolution of morphological innovations but instead exhibit a pronounced correlation with movement into new geographic areas, particularly the dispersal of lineages into new mountainous regions. The interdependence among apparent novelties (arising from their nested phylogenetic distribution) and the correlation between morphological and biogeographic change suggests a complex history of diversification in Dipsacales. Overall, these findings highlight the importance of incorporating biogeographic history in studies of diversification rates and in the study of geographic gradients in species richness. Furthermore, these results argue against a simple deterministic relationship between dispersal and diversification: like other factors that may influence the probability of speciation and/or extinction, the impact of dispersal on diversification rates depends on being in the right place at the right time.

Preliminary phylogeny of Valerianaceae (Dipsacales) inferred from nuclear and chloroplast DNA sequence data.

Valerianaceae is a relatively small (ca. 350 species), but morphologically diverse angiosperm clade. Sequence data from the entire ndhF gene, the trnL-F intergenic spacer region, the trnL intron, the matK region, the rbcL-atpB intergenic spacer region and internal transcribed spacer (ITS) region of nuclear ribosomal DNA were collected for 21 taxa within Dipsacaceae and Valerianaceae (1 and 20, respectively). These data were included in several phylogenetic analyses with previously published sequences from Dipsacales. Results from these analyses (maximum parsimony, maximum likelihood, and Bayesian analysis) are in strong agreement with many of the conclusions from previous studies, most importantly: (1) Valerianaceae is sister to Dipsacaceae; (2) Triplostegia is more closely related to species of Dipsacaceae than to Valerianaceae; and (3) Valeriana appears not to be monophyletic, with Valeriana celtica falling outside the remainder of the species of Valeriana sampled here (with very strong support). With the exception of V. celtica, these data support two major clades within Valeriana; one that is exclusively New World and another that is distributed in both the Old and New World. Although the species of Valerianaceae and its sister group Dipsacaceae plus Triplostegia, are widely distributed in the Northern Hemisphere, and the data imply that Valerianaceae diversified initially in Asia (the Himalayan Patrinia and Nardostachys falling at the base of the clade), the center of modern species diversity for the group is in the Andes of South America with as many as 175 species restricted to that region. Although the exclusively South American taxa form a clade in the chloroplast and combined ITS and chloroplast analyses, support values tend to be low. Future studies will need to include additional data, in the form of both characters and taxa, before any strong conclusions about the character evolution, diversification, and biogeography of the South American valerians can be made.

Phylogeny of Valerianaceae based on matK and ITS markers, with reference to matK individual polymorphism.

BACKGROUND AND AIMS: The monophyly of Valerianaceae and the precise delimitation of the family are not totally resolved. Our knowledge on the phylogeny of the group is only partial: on a morphological basis, some contradicting taxonomic proposals have been published, which demonstrates the difficulties in establishing a natural classification of the family and especially in proposing a relevant treatment of the large genus Valeriana. The aims of this study are to contribute to the phylogeny and generic delineation of the Valerianaceae on the basis of molecular data. METHODS: A cladistic analysis of the sequences of one plastid (matK) and one nuclear (ITS) molecular marker was carried out, both individually and in combination. KEY RESULTS: The results of the analyses of both regions confirm that the family is monophyletic, with the exclusion of Triplostegia. The tribe Patrinieae is monophyletic, and the tribe Valerianeae is also a natural group. Two of the subtribes of Valerianeae, Fediinae and Centranthinae, are also monophyletic, with the exclusion of the genus Plectritis from Fediinae. The subtribe Valerianinae, on the other hand, is paraphyletic. CONCLUSIONS: Our results confirm, for the first time on a molecular basis, the suggested paraphyly of Valeriana in its present circumscription, with profound nomenclatural and taxonomic implications. The correlation between molecular phylogeny and biogeography is close. In the course of the plastid DNA sequencing, a polymorphism concerning the matK gene was found, a fact that should be carefully evaluated in phylogenetic analyses.

Coding of insertion-deletion events of the chloroplastic intergene atp beta-rbcL for the phylogeny of the Valerianeae tribe (Valerianaceae).

A preliminary analysis of the sequence alignment of the chloroplast intergene atp beta-rbcL in tribe Valerianeae reveals that insertion-deletion evolutionary events ('indels'), combined with nucleotide substitutions, have occurred in large zones in some of the studied taxa. Due to the frequent occurrence and large size of indels within this tribe, intergene length varies from 531 to 788 base pairs within the studied species. This situation poses gap coding problems that we had to tackle before phylogenetic analysis. Four methods of gap coding were used: elimination of gapped sites ('complete omission'), 'missing data', 'fifth base' and Barriel's coding method, which translates indels into new multistate characters in the data matrix. After application of these four methods of data treatment, phylogenetic analyses (maximum parsimony) did not lead to very different results. Three robust clades emerged in each case, corresponding to the Centranthinae subtribe (genus Centranthus), the Fediinae subtribe (genera Fedia and Valerianella), and the American species of Valeriana. The theoretical basis and biological significance of these four methods are discussed in order to apply the best ones in future studies.