Chun-Neng Wang

First insights into fern matK phylogeny.

MatK, the only maturase gene in the land plant plastid genome, is a very popular phylogenetic marker that has been extensively applied in reconstructing angiosperm phylogeny. However, the use of matK in fern phylogeny is largely unknown, due to difficulties with amplification: ferns have lost the flanking trnK exons, typically the region used for designing stable priming sites. We developed primers that are either universal or lineage-specific that successfully amplify matK across all fern families. To evaluate whether matK is as powerful a phylogenetic marker in ferns as in angiosperms, we compared its sequence characteristics and phylogenetic performance to those of rbcL and atpA. Among these three genes, matK has the highest variability and substitution evenness, yet shows the least homoplasy. Most importantly, applying matK in fern phylogenetics better resolved relationships among families, especially within eupolypods I and II. Here we demonstrate the power of matK for fern phylogenetic reconstruction, as well as provide primers and extensive sequence data that will greatly facilitate future evolutionary studies of ferns.

Flower development of Phalaenopsis orchid involves functionally divergent SEPALLATA-like genes

The Phalaenopsis orchid produces complex flowers that are commercially valuable, which has promoted the study of its flower development. E-class MADS-box genes, SEPALLATA (SEP), combined with B-, C- and D-class MADS-box genes, are involved in various aspects of plant development, such as floral meristem determination, organ identity, fruit maturation, seed formation and plant architecture. Four SEP-like genes were cloned from Phalaenopsis orchid, and the duplicated PeSEPs were grouped into PeSEP1/3 and PeSEP2/4. All PeSEPs were expressed in all floral organs. PeSEP2 expression was detectable in vegetative tissues. The study of protein–protein interactions suggested that PeSEPs may form higher order complexes with the B-, C-, D-class and AGAMOUS LIKE6-related MADS-box proteins to determine floral organ identity. The tepal became a leaf-like organ when PeSEP3 was silenced by virus-induced silencing, with alterations in epidermis identity and contents of anthocyanin and chlorophyll. Silencing of PeSEP2 had minor effects on the floral phenotype. Silencing of the E-class genes PeSEP2 and PeSEP3 resulted in the downregulation of B-class PeMADS2-6 genes, which indicates an association of PeSEP functions and B-class gene expression. These findings reveal the important roles of PeSEP in Phalaenopsis floral organ formation throughout the developmental process by the formation of various multiple protein complexes.

Phylogenetic Position of Titanotrichum oldhamii (Gesneriaceae) Inferred From Four Different Gene Regions

Abstract Titanotrichum oldhamii has been variously placed in Gesneriaceae or Scrophulariaceae, although most recent taxonomic treatments treat it as a monotypic tribe within Gesneriaceae. In this study, we reconstructed a broad-scale phylogeny containing Titanotrichum using gene sequences from four sequence regions (chloroplast trnL-F intron and spacer and atpB-rbcL spacer, nuclear 26S ribosomal DNA, and the low-copy developmental gene CYCLOIDEA, CYC). The phylogenies inferred from each individual data set and the combined data are congruent in placing Titanotrichum inside Gesneriaceae. The phylogenetic tree based on combined chloroplast and nuclear DNA sequences grouped Titanotrichum with subfamilies Gesnerioideae (New World) and Coronantheroideae (South Pacific and Chile). We have isolated CYC, from most of the species of Gesneriaceae and Scrophulariaceae represented in this study, and this gene phylogeny suggests the same placement of Titanotrichum. CYC was found to evolve three times faster than the trnL-F intron and spacer, 3.3 times faster than the atpB-rbcL spacer, and eight times faster than nuclear 26S rDNA. Although there is considerable phylogenetic information in this fast evolving gene, analysis is problematic because of high levels of homoplasy and paralogy. In addition to a duplication predating a split between New World and Old World taxa (Gcyc1 vs. Gcyc2), there are several subsequent lineage-related duplications (mainly within Gcyc1).

Meristem fate and bulbil formation in Titanotrichum (Gesneriaceae)

Titanotrichum oldhamii (a monotypic genus from Taiwan, Okinawa, and adjacent regions of China) has inflorescences bearing either showy yellow flowers or asexual bulbils. Asexual reproduction by bulbils is important in natural populations, and bulbil production increases in August and September at the end of the flowering season (which runs from June to the end of September). The bulbils are small (∼1-2.5 mm long) and numerous. They consist of a small portion of stem (bract-stem) topped by opposite storage bracts that enclose a minute apical meristem. A secondary root develops from the side of the bract-stem. The floral meristem of T. oldhamii has three possible fates: (1) bulbil formation, (2) flower formation, or (3) bracteose proliferation. Bracteose proliferation rarely occurs and appears to be a developmental transition between the bulbiliferous and racemose inflorescence forms. It is strongly reminiscent of the floricaula and squamosa mutants of Antirrhinum. In the bulbiliferous form a single floral primordium, which would normally produce one flower, gives rise to ∼50-70 bulbils by repeated subdivision of the meristem. This form of bulbil production appears to be unique to Titanotrichum. Occasionally a floral meristem divides, but the subdivision forms multiflowered units of up to four flowers rather than bulbils, suggesting that meristem fate is reversible up to the first or second meristem subdivision. In Titanotrichum, therefore, primordium fate is apparently not determined at inception but becomes irreversibly determined shortly after the appearance of developmental characteristics of the floral or bulbil pathway.

Molecular population genetics and gene expression analysis of duplicated CBF genes of Arabidopsis thaliana

BackgroundCBF/DREB duplicate genes are widely distributed in higher plants and encode transcriptional factors, or CBFs, which bind a DNA regulatory element and impart responsiveness to low temperatures and dehydration.ResultsWe explored patterns of genetic variations of CBF1, -2, and -3 from 34 accessions of Arabidopsis thaliana. Molecular population genetic analyses of these genes indicated that CBF2 has much reduced nucleotide diversity in the transcriptional unit and promoter, suggesting that CBF2 has been subjected to a recent adaptive sweep, which agrees with reports of a regulatory protein of CBF2. Investigating the ratios of Ka/Ks between all paired CBF paralogus genes, high conservation of the AP2 domain was observed, and the major divergence of proteins was the result of relaxation in two regions within the transcriptional activation domain which was under positive selection after CBF duplication. With respect to the level of CBF gene expression, several mutated nucleotides in the promoters of CBF3 and -1 of specific ecotypes might be responsible for its consistently low expression.ConclusionWe concluded from our data that important evolutionary changes in CBF1, -2, and -3 may have primarily occurred at the level of gene regulation as well as in protein function.

Identifying a mysterious aquatic fern gametophyte

Süßwassertang, a popular aquatic plant that is sold worldwide in aquarium markets, has been long considered a liverwort because of its ribbon-like thallus. However, its antheridia are remarkably fern-like in morphology. To corroborate the hypothesis that Süßwassertang is a fern gametophyte and to determine its closest relative, we have sequenced five chloroplast regions (rbcL, accD, rps4–trnS, trnL intron, and trnL-F intergenic spacer), applying a DNA-based identification approach. The BLAST results on all regions revealed that Süßwassertang is a polypod fern (order: Polypodiales) with strong affinities to the Lomariopsidaceae. Our phylogenetic analyses further showed that Süßwassertang is nested within the hemi-epiphytic fern genus Lomariopsis (Lomariopsidaceae) and aligned very close to L. lineata. Our study brings new insights on the unexpected biology of Lomariopsis gametophytes—the capacity of retaining a prolonged gametophytic stage under water. It is of great interest to discover that a fern usually known to grow on trees also has gametophytes that thrive in water.

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

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

MicroRNA396-Targeted SHORT VEGETATIVE PHASE Is Required to Repress Flowering and Is Related to the Development of Abnormal Flower Symptoms by the Phyllody Symptoms1 Effector

A PHYL1 effector protein interferes with miR396-mediated transcriptional regulator mRNA decay, enhancing the transcription factor for abnormal flower development. Leafy flowers are the major symptoms of peanut witches’ broom (PnWB) phytoplasma infection in Catharanthus roseus. The orthologs of the phyllody symptoms1 (PHYL1) effector of PnWB from other species of phytoplasma can trigger the proteasomal degradation of several MADS box transcription factors, resulting in leafy flower formation. In contrast, the flowering negative regulator gene SHORT VEGETATIVE PHASE (SVP) was up-regulated in PnWB-infected C. roseus plants, but most microRNA (miRNA) genes had repressed expression. Coincidentally, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the PHYL1 gene of PnWB (PHYL1 plants), which show leafy flower phenotypes, up-regulate SVP of Arabidopsis (AtSVP) but repress a putative regulatory miRNA of AtSVP, miR396. However, the mechanism by which PHYL1 regulates AtSVP and miR396 is unknown, and the evidence of miR396-mediated AtSVP degradation is lacking. Here, we show that miR396 triggers AtSVP messenger RNA (mRNA) decay using genetic approaches, a reporter assay, and high-throughput degradome profiles. Genetic evidence indicates that PHYL1 plants and atmir396a-1 mutants have higher AtSVP accumulation, whereas the transgenic plants overexpressing MIR396 display lower AtSVP expression. The reporter assay indicated that target-site mutation results in decreasing the miR396-mediated repression efficiency. Moreover, degradome profiles revealed that miR396 triggers AtSVP mRNA decay rather than miRNA-mediated cleavage, implying that AtSVP caused miR396-mediated translation inhibition. We hypothesize that PHYL1 directly or indirectly interferes with miR396-mediated AtSVP mRNA decay and synergizes with other effects (e.g. MADS box transcription factor degradation), resulting in abnormal flower formation. We anticipate our findings to be a starting point for studying the posttranscriptional regulation of PHYL1 effectors in symptom development.

Quantifying floral shape variation in 3D using microcomputed tomography: a case study of a hybrid line between actinomorphic and zygomorphic flowers

The quantification of floral shape variations is difficult because flower structures are both diverse and complex. Traditionally, floral shape variations are quantified using the qualitative and linear measurements of two-dimensional (2D) images. The 2D images cannot adequately describe flower structures, and thus lead to unsatisfactory discrimination of the flower shape. This study aimed to acquire three-dimensional (3D) images by using microcomputed tomography (μCT) and to examine the floral shape variations by using geometric morphometrics (GM). To demonstrate the advantages of the 3D-μCT-GM approach, we applied the approach to a second-generation population of florists gloxinia (Sinningia speciosa) crossed from parents of zygomorphic and actinomorphic flowers. The flowers in the population considerably vary in size and shape, thereby served as good materials to test the applicability of the proposed phenotyping approach. Procedures were developed to acquire 3D volumetric flower images using a μCT scanner, to segment the flower regions from the background, and to select homologous characteristic points (i.e., landmarks) from the flower images for the subsequent GM analysis. The procedures identified 95 landmarks for each flower and thus improved the capability of describing and illustrating the flower shapes, compared with typically lower number of landmarks in 2D analyses. The GM analysis demonstrated that flower opening and dorsoventral symmetry were the principal shape variations of the flowers. The degrees of flower opening and corolla asymmetry were then subsequently quantified directly from the 3D flower images. The 3D-μCT-GM approach revealed shape variations that could not be identified using typical 2D approaches and accurately quantified the flower traits that presented a challenge in 2D images. The approach opens new avenues to investigate floral shape variations.

Tissue‐direct PCR, a rapid and extraction‐free method for barcoding of ferns

Fern gametophytes and young sporophytes often provide too little material for DNA extraction and are particularly difficult to identify to genus. Here we developed an efficient procedure called ‘Tissue‐direct PCR’, in which a slice of fern tissue is mixed with PCR reagents and primers, allowing certain genomic regions to be amplified directly in the thermal cycler. For these diminutive and featureless stages of ferns, Tissue‐direct PCR combined with amplifying plant barcodes promises to make the identification of immature ferns easy and rapid. Tissue‐direct PCR would also be very helpful for large‐scale ecological studies surveying distribution and population structure.