Molecular phylogeny of Asian Ardisia (Myrsinoideae, Primulaceae) and their leaf-nodulated endosymbionts, Burkholderia s.l. (Burkholderiaceae).

The genus Ardisia (Myrsinoideae, Primulaceae) has 16 subgenera and over 700 accepted names, mainly distributed in tropical Asia and America. The circumscription of Ardisia is not well-defined and sometimes confounded with the separation of some small genera. A taxonomic revision focusing on Ardisia and allies is necessary. In the Ardisia subgenus Crispardisia, symbiotic association with leaf-nodule bacteria is a unique character within the genus. The endosymbionts are vertically transmitted, highly specific and highly dependent on the hosts, suggesting strict cospeciation may have occurred in the evolutionary history. In the present study, we aimed to establish a phylogenetic framework for further taxonomic revision. We also aimed to test the cospeciation hypothesis of the leaf-nodulate Ardisia and their endosymbiotic bacteria. Nuclear ITS and two chloroplast intergenic spaces were used to reconstruct the phylogeny of Asian Ardisia and relatives in Myrsinoideae, Primulaceae. The 16S-23S rRNA were used to reconstruct the bacterial symbionts' phylogeny. To understand the evolutionary association of the Ardisia and symbionts, topology tests and cophylogenetic analyses were conducted. The molecular phylogeny suggested Ardisia is not monophyletic, unless Sardiria, Hymenandra, Badula and Oncostemum are included. The results suggest the generic limit within Myrsinoideae (Primulaceae) needs to be further revised. The subgenera Crispardisia, Pimelandra, and Stylardisia were supported as monophyly, while the subgenus Bladhia was separated into two distant clades. We proposed to divide the subgenus Bladhia into subgenus Bladhia s.str. and subgenus Odontophylla. Both of the cophylogenetic analyses and topology tests rejected strict cospeciation hypothesis between Ardisia hosts and symbiotic Burkholderia. Cophylogenetic analyses showed general phylogenetic concordance of Ardisia and Burkholderia, and cospeciation events, host-switching events and loss events were all inferred.

DCL2- and RDR6-dependent transitive silencing of SMXL4 and SMXL5 in Arabidopsis dcl4 mutants causes defective phloem transport and carbohydrate over-accumulation.

DICER-LIKE (DCL) enzymes process double-stranded RNA into small RNAs that act as regulators of gene expression. Arabidopsis DCL4 and DCL2 each allow the post-transcriptional gene silencing (PTGS) of viruses and transgenes, but primary PTGS-prone DCL4 outcompetes transitive PTGS-prone DCL2 in wild-type plants. This hierarchy likely prevents DCL2 having any detrimental effects on endogenous genes. Indeed, dcl4 mutants exhibit developmental defects and increased sensitivity to genotoxic stress. In this study, the mechanism underlying dcl4 defects was investigated using genetic, biochemical and high-throughput sequencing approaches. We show that the purple phenotype of dcl4 leaves correlates with carbohydrate over-accumulation and defective phloem transport, and depends on the activity of SUPPRESSOR OF GENE SILENCING 3, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DCL2. This phenotype correlates with the downregulation of two genes expressed in the apex and the vasculature, SMAX1-LIKE 4 (SMXL4) and SMXL5, and the accumulation of DCL2- and RDR6-dependent small interfering RNAs derived from these two genes. Supporting a causal effect, smxl4 smxl5 double mutants exhibit leaf pigmentation, enhanced starch accumulation and defective phloem transport, similar to dcl4 plants. Overall, this study elucidates the detrimental action of DCL2 when DCL4 is absent, and indicates that DCL4 outcompeting DCL2 in wild-type plants is crucial to prevent the degradation of endogenous transcripts by DCL2- and RDR6-dependent transitive PTGS.

Concomitant loss of NDH complex-related genes within chloroplast and nuclear genomes in some orchids.

The chloroplast NAD(P)H dehydrogenase-like (NDH) complex consists of about 30 subunits from both the nuclear and chloroplast genomes and is ubiquitous across most land plants. In some orchids, such as Phalaenopsis equestris, Dendrobium officinale and Dendrobium catenatum, most of the 11 chloroplast genome-encoded ndh genes (cp-ndh) have been lost. Here we investigated whether functional cp-ndh genes have been completely lost in these orchids or whether they have been transferred and retained in the nuclear genome. Further, we assessed whether both cp-ndh genes and nucleus-encoded NDH-related genes can be lost, resulting in the absence of the NDH complex. Comparative analyses of the genome of Apostasia odorata, an orchid species with a complete complement of cp-ndh genes which represents the sister lineage to all other orchids, and three published orchid genome sequences for P. equestris, D. officinale and D. catenatum, which are all missing cp-ndh genes, indicated that copies of cp-ndh genes are not present in any of these four nuclear genomes. This observation suggests that the NDH complex is not necessary for some plants. Comparative genomic/transcriptomic analyses of currently available plastid genome sequences and nuclear transcriptome data showed that 47 out of 660 photoautotrophic plants and all the heterotrophic plants are missing plastid-encoded cp-ndh genes and exhibit no evidence for maintenance of a functional NDH complex. Our data indicate that the NDH complex can be lost in photoautotrophic plant species. Further, the loss of the NDH complex may increase the probability of transition from a photoautotrophic to a heterotrophic life history.

Homalium glandulosum (Salicaceae), a new species from Vu Quang National Park, North Central Vietnam.

Homalium glandulosum Tagane & V. H. Nguyen, from Vu Quang National Park in northern Vietnam, is newly described. This species is characterized by distinct glands, often stalked, at the base of the lamina and along the margin of the stipules and bracteoles. Illustrations, DNA barcodes of the two regions of rbcL and matK, and a key to the species of Homalium in Vietnam are also provided.

Asymmetric bulges and mismatches determine 20-nt microRNA formation in plants.

Plant microRNAs (miRNAs) are predominantly 21 nucleotides (nt) long but non-canonical lengths of 22 and 20 nt are commonly observed in diverse plant species. While miRNAs longer than 21 nt can be attributed to the neglect of unpaired bases within asymmetric bulges by the ruler function of dicer-like 1 (DCL1), how 20-nt miRNA is generated remains obscure. Analysis of small RNA data revealed that 20-nt miRNA can be divided into 3 main groups featured by atypical 3' overhangs or shorter duplex regions. Asymmetric bulges or mismatches at specific positions are commonly observed within each group and were shown to be crucial for 20-nt miRNA formation. Analysis of DCL1 cleavage sites on 20-nt miRNA precursors suggests that these determinants might alter precursor structure or trigger 3'-end decay of mature miRNA. The results herein advance our understanding of miRNA biogenesis and demonstrate that the effect of asymmetric bulges on miRNA length could be position-dependent.