Gene duplication allows for functional divergence and innovation that provide selective advantages. However, in flowering plants genetic studies have revealed that single-gene mutations affecting one of two or more closely related paralogs often fail to cause detectable morphological defects, suggesting functional redundancy. Flowering plants have hundreds of genes encoding leucine-rich repeat receptor-like protein kinases (LRR-RLKs), several of which play important roles in anther development, but little is known about their evolutionary history and possible functional divergence. We investigated the evolutionary relationship of the LRR-RLK gene family by phylogenetic analysis and found that these closely related paralogs resulted from multiple duplication events, such as the one resulting in BAM1 and BAM2. We further used quantitative real-time reverse transcription–polymerase chain reaction to verify gene expression changes in immature anthers from the bam1/bam2single and double mutants compared with wild type, providing strong evidence that the BAM1 and BAM2 genes have evolved different functions, with differential effects on anther gene expression. Moreover, careful examination of anther development in bam1 and bam2 single mutants revealed previously unrecognized extra cell division in tapetum cell layers. Thus our results from phylogenetic, molecular, and morphological analyses uncover sequence and functional differences between paralogs whose single mutants lack obvious fertility defects, effectively revealing functional divergence of duplicate genes.

Chloroplast genome information helps improve the phylogenetic resolution and can act as organelle-scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long-range polymerase chain reaction and using next-generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel-dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel-dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.

Distinguishing yam species based on morphological traits is extremely difficult and unreliable, posing a challenge to breeders and genebank curators. Development of a molecular assay based on DNA barcoding can facilitate rapid and accurate identification of important Dioscorea species. To develop a DNA barcoding system forDioscorea species identification, the rbcL and matK loci (in unison and in combination), the non-coding intergenic spacer trnH-psbA of the chloroplast genome, and the nuclear ITS regions were investigated using criteria for developing candidate DNA barcodes. All DNA barcoding sequences were assessed for ease of PCR amplification, sequence quality and species discriminatory power. Amongst the markers investigated, the matK locus performed well in terms of species identification (63.2%), in addition to detecting high interspecific variation with mean divergence of 0.0196 (SD=0.0209). The combination of the two coding regions (rbcL + matK) was determined to be the optimal (76.2%) DNA barcoding approach as 16 out of 21 species could be defined. While the rbcL exhibited good PCR amplification efficiency and sequence quality, its species discriminatory power was relatively poor with 47.6% identification. Similarly, the trnH-psbA region had a weak discrimination efficiency of only 36.8%. While the development of more robust DNA barcoding systems is an ongoing challenge, our results indicate that therbcL + matK combination can be utilized as multi-locus DNA barcode regions for Dioscorea species identification.

Low copy nuclear genes show great potential to provide phylogenetic information, but their use has been hampered by several inherent adverse factors. Polymerase chain reaction (PCR)-mediated recombination ranks among these factors and occurs when high levels of similar paralogs for a low-copy nuclear gene coexist within a single PCR amplification reaction. In this study, the LEAFY gene was cloned and sequenced for 63 Cinnamomum species and two copies of the second intron were found within species of the diploid sect. Cinnamomum. Although these two copies are very similar, they can be distinguished easily due to a specific ca. 47-bp segment that is missing in the “short sequence” copy. The “long sequence” copy performed well in phylogenetic analysis of Cinnamomum and is largely consistent with phylogenetic relationships based on internal transcribed spacer region sequences. In contrast, the “short sequence” copy was problematic for phylogenetic reconstruction and PCR-mediated recombination was detected in 20 of the 38 Cinnamomum species with two LEAFY copies. Thirty-one recombinants were discriminated and the breakpoints suggested by the programs RDP3 and GARD were distributed randomly along the recombination sequences. This study shows that duplication in low-copy nuclear genes and problems associated with PCR-mediated recombination need to be given more attention in phylogenetic studies.

The level and pattern of nucleotide variation in duplicate genes provide important information on the evolutionary history of polyploids and divergent processes between homoeologous loci within lineages. Leymus, a group of allopolyploid species with the NsXm genomes, is a perennial genus with a diverse array of morphology, ecology, and distribution in Triticeae. To estimate the phylogeny and molecular evolution of a single-copy DMC1 gene in Leymus and its diploid relatives,DMC1 homoeologous sequences were isolated from the sampled Leymus species and were analyzed with those from 30 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that: (i) different Leymus species might derive their Ns genome from different Psathyrostachys species; (ii) Pseudoroegneria has contributed to the nuclear genome of some Leymus species, which might result from recurrent hybridization or incomplete lineage sorting; (iii) the Xm genome origin of Leymus could differ among species; (iv) rapid radiation and multiple origin might account for the rich diversity, numbers of species, and wide ecological adaptation of Leymus species; and (v) the DMC1 sequence diversity of the Ns genome in Leymus species was lower than that in the Psathyrostachys diploids, while the level of DMC1 sequence diversity in Leymus was higher than that in diploid Pseudoroegneria. Our results provide new insight on the evolutionary dynamics of duplicate DMC1 genes, polyploid speciation, and the phylogeny of Leymus species.

Today southern Yunnan, SW China, has a tropical or subtropical climate and seasonal rainforests. In the past, some temperate elements were also present. In this paper, a new species of Populus is reported from the Middle Miocene deposits in Zhenyuan. Its leaves are ovate or ovate-suborbicular, with serrate margins. They have a shallowly cordate to cuneate base without glands, short acuminate apex, and salicoid teeth with spherical glands. The veins are glabrous but unicellular hair bases occur on the lower epidermis of the lamina. Stomata are confined to the lower epidermis. The presence of Populus in the Middle Miocene of the region indicates an expansion of the genus into low-latitude Asia in the late Cenozoic and a more complicated history of vegetational change in southern Yunnan than has so far been assumed.