Revisiting chloroplast genomic landscape and annotation towards comparative chloroplast genomes of Rhamnaceae.

BACKGROUND: Massive parallel sequencing technologies have enabled the elucidation of plant phylogenetic relationships from chloroplast genomes at a high pace. These include members of the family Rhamnaceae. The current Rhamnaceae phylogenetic tree is from 13 out of 24 Rhamnaceae chloroplast genomes, and only one chloroplast genome of the genus Ventilago is available. Hence, the phylogenetic relationships in Rhamnaceae remain incomplete, and more representative species are needed. RESULTS: The complete chloroplast genome of Ventilago harmandiana Pierre was outlined using a hybrid assembly of long- and short-read technologies. The accuracy and validity of the final genome were confirmed with PCR amplifications and investigation of coverage depth. Sanger sequencing was used to correct for differences in lengths and nucleotide bases between inverted repeats because of the homopolymers. The phylogenetic trees reconstructed using prevalent methods for phylogenetic inference were topologically similar. The clustering based on codon usage was congruent with the molecular phylogenetic tree. The groups of genera in each tribe were in accordance with tribal classification based on molecular markers. We resolved the phylogenetic relationships among six Hovenia species, three Rhamnus species, and two Ventilago species. Our reconstructed tree provides the most complete and reliable low-level taxonomy to date for the family Rhamnaceae. Similar to other higher plants, the RNA editing mostly resulted in converting serine to leucine. Besides, most genes were subjected to purifying selection. Annotation anomalies, including indel calling errors, unaligned open reading frames of the same gene, inconsistent prediction of intergenic regions, and misannotated genes, were identified in the published chloroplast genomes used in this study. These could be a result of the usual imperfections in computational tools, and/or existing errors in reference genomes. Importantly, these are points of concern with regards to utilizing published chloroplast genomes for comparative genomic analysis. CONCLUSIONS: In summary, we successfully demonstrated the use of comprehensive genomic data, including DNA and amino acid sequences, to build a reliable and high-resolution phylogenetic tree for the family Rhamnaceae. Additionally, our study indicates that the revision of genome annotation before comparative genomic analyses is necessary to prevent the propagation of errors and complications in downstream analysis and interpretation.

Historical biogeography of Pomaderris (Rhamnaceae): Continental vicariance in Australia and repeated independent dispersals to New Zealand.

AIM: Gondwanan biogeographic patterns include a combination of old vicariance events following the breakup of the supercontinent, and more recent long-distance dispersals across the southern landmasses. Floristic relationships between Australia and New Zealand have mostly been attributed to recent dispersal events rather than vicariance. We assessed the biogeographic history of Pomaderris (Rhamnaceae), which occurs in both Australia and New Zealand, by constructing a time-calibrated molecular phylogeny to infer (1) phylogenetic relationships and (2) the relative contributions of vicariance and dispersal events in the biogeographic history of the genus. LOCATION: Australia and New Zealand. METHODS: Using hybrid capture and high throughput sequencing, we generated nuclear and plastid data sets to estimate phylogenetic relationships and fossil calibrated divergence time estimates for Pomaderris. BioGeoBEARS and biogeographical stochastic mapping (BSM) were used to assess the ancestral area of the genus and the relative contributions of vicariance vs dispersal, and the directionality of dispersal events. RESULTS: Our analyses indicate that Pomaderris originated in the Oligocene and had a widespread Australian distribution. Vicariance of western and eastern Australian clades coincides with the uplift of the Nullarbor Plain c. 14 Ma, followed by subsequent in-situ and within-biome diversification with little exchange across regions. A rapid radiation of southeastern Australian taxa beginning c. 10 Ma was the source for at least six independent long-distance dispersal events to New Zealand during the Pliocene-Pleistocene. MAIN CONCLUSIONS: Our study demonstrates the importance of dispersal in explaining not only the current cross-Tasman distributions of Pomaderris, but for the New Zealand flora more broadly. The pattern of multiple independent long-distance dispersal events for Pomaderris, without significant radiation within New Zealand, is congruent with other lowland plant groups, suggesting that this biome has a different evolutionary history compared with the younger alpine flora of New Zealand, which exhibits extensive radiations often following single long distance dispersal events.

The Peptidoglycan Biosynthesis Gene murC in Frankia: Actinorhizal vs. Plant Type.

Nitrogen-fixing Actinobacteria of the genus Frankia can be subdivided into four phylogenetically distinct clades; members of clusters one to three engage in nitrogen-fixing root nodule symbioses with actinorhizal plants. Mur enzymes are responsible for the biosynthesis of the peptidoglycan layer of bacteria. The four Mur ligases,MurC, MurD, MurE, and MurF, catalyse the addition of a short polypeptide to UDP-N-acetylmuramic acid. Frankia strains of cluster-2 and cluster-3 contain two copies of murC, while the strains of cluster-1 and cluster-4 contain only one. Phylogenetically, the protein encoded by the murC gene shared only by cluster-2 and cluster-3, termed MurC1, groups with MurC proteins of other Actinobacteria. The protein encoded by the murC gene found in all Frankia strains, MurC2, shows a higher similarity to the MurC proteins of plants than of Actinobacteria. MurC2 could have been either acquired via horizontal gene transfer or via gene duplication and convergent evolution, while murC1 was subsequently lost in the cluster-1 and cluster-4 strains. In the nodules induced by the cluster-2 strains, the expression levels of murC2 were significantly higher than those of murC1. Thus, there is clear sequence divergence between both types of Frankia MurC, and Frankia murC1 is in the process of being replaced by murC2, indicating selection in favour of murC2. Nevertheless, protein modelling showed no major structural differences between the MurCs from any phylogenetic group examined.

Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization.

Nitrogen-fixing filamentous Frankia colonize the root tissues of its actinorhizal host Discaria trinervis via an exclusively intercellular pathway. Here we present studies aimed at uncovering mechanisms associated with this little-researched mode of root entry, and in particular the extent to which the host plant is an active partner during this process. Detailed characterization of the expression patterns of infection-associated actinorhizal host genes has provided valuable tools to identify intercellular infection sites, thus allowing in vivo confocal microscopic studies of the early stages of Frankia colonization. The subtilisin-like serine protease gene Dt12, as well as its Casuarina glauca homolog Cg12, are specifically expressed at sites of Frankia intercellular colonization of D. trinervis outer root tissues. This is accompanied by nucleo-cytoplasmic reorganization in the adjacent host cells and major remodeling of the intercellular apoplastic compartment. These findings lead us to propose that the actinorhizal host plays a major role in modifying both the size and composition of the intercellular apoplast in order to accommodate the filamentous microsymbiont. The implications of these findings are discussed in the light of the analogies that can be made with the orchestrating role of host legumes during intracellular root hair colonization by nitrogen-fixing rhizobia.

Genome-wide identification and characterization of the SPL gene family in Ziziphus jujuba.

SQUAMOSA Promoter-Binding Protein-Likes (SPLs) are plant specific transcription factors playing important roles in plant growth and development. The SPL gene family has been studied in various plant species; however, there is no report about SPLs in Zizyphus jujuba. In this study, we identified 18 putative ZjSPL genes in Z. jujuba using a genome-wide analysis. Sequence features, gene structures, conserved domains and motifs were analyzed. The phylogenetic relationships of SPLs in Z. jujuba and A. thaliana were revealed. A total of 5 pairs of ZjSPLs were identified, suggesting the importance of gene duplication in SPL gene expansion in Z. jujuba. In addition, 11 of the 18 ZjSPLs, belonging to G1, G2 and G5 subgroups, were found to be targets of miR156, suggesting the conservation of miR156-mediated posttranscriptional regulation in plants. Expression analysis revealed that eight ZjSPL genes were responsive to the infection of witches'-broom phytoplasma. Our results provide a basis for the further elucidation of the biological function of ZjSPLs and their regulation in witches'-broom disease.

Microsatellite markers from Ceanothus roderickii (Rhamnaceae) using next-generation sequencing technology.

PREMISE OF THE STUDY: Ceanothus roderickii is an endangered shrub endemic to California. To investigate the population genetics of this species, including the genetic consequences of population fragmentation and hybridization, 10 microsatellite markers were developed. METHODS AND RESULTS: Using next-generation sequencing (454) data from a single C. roderickii individual, 10 microsatellite markers were developed. A group of 12 individuals representing all of the major C. roderickii populations were analyzed. All loci were found to be polymorphic, with a range from two to 12 alleles per locus. Observed heterozygosity ranged from 0.08 to 0.83 across loci. All 10 loci were also amplifiable in at least one other Ceanothus species. CONCLUSIONS: Results presented here indicate the utility of our new microsatellite primers in ongoing and future studies concerning population genetics and gene flow in C. roderickii, as well as the potential applicability of these primers in similar studies on other Ceanothus species.

Genetic diversity in fragmented populations of Berchemiella wilsonii var. pubipetiolata (Rhamnaceae).

BACKGROUND AND AIMS: [corrected] Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is distributed in fragmented habitat patches in eastern China. It is highly endangered because of severe disturbance by anthropogenic activities. Information on genetic variation and structure is critical for developing successful conservation strategies for this species. METHODS: Allozyme variation of population genetic diversity and structure was investigated for a total of 98 individuals sampled from four extant populations using isoelectric focusing in thin-layer polyacrylamide slab gels. KEY RESULTS: Based on 20 loci scored from the nine enzymes examined, a high genetic diversity was detected at both the species and population level, while there was a loss of low frequency alleles (<0.1) in all populations. Most loci showed deviation from Hardy-Weinberg equilibrium due to excess of heterozygotes in all populations, suggesting that selection for heterozygotes has occurred in this species. The genetic diversity was mainly found within populations with a moderate genetic differentiation (F(ST) = 0.13), but the two geographically discontinuous population groups showed significant differences, with F-statistic values of 0.078 for the Zhejiang populations and 0.014 for the Anhui populations, respectively. CONCLUSIONS: It appears most likely that this species has experienced a recent decrease in population size, and genetic drift in small populations has resulted in a loss of alleles occurring at low frequency. The differentiation into two population groups reflects a population genetic consequence that has been influenced by the different land-use in the two regions. Some conservation concerns are discussed together with possible strategies for implementing in situ and ex situ conservation.

Historical biogeography of two cosmopolitan families of flowering plants: Annonaceae and Rhamnaceae.

Annonaceae are a pantropically distributed family found predominantly in rainforests, so they are megathermal taxa, whereas Rhamnaceae are a cosmopolitan family that tend to be found in xeric regions and may be classified as mesothermal. Phylogenetic analyses of these families are presented based on rbcL and trnL-F plastid DNA sequences. Likelihood ratio tests revealed rate heterogeneity in both phylogenetic trees and they were therefore made ultrametric using non-parametric rate smoothing and penalized likelihood. Divergence times were then estimated using fossil calibration points. The historical biogeography of these families that are species rich in different biomes is discussed and compared with other published reconstructions. Rhamnaceae and most lineages within Annonaceae are too young to have had their distribution patterns influenced by break-up of previously connected Gondwanan landmasses. Contrasts in the degree of geographical structure between these two families may be explained by differences in age and dispersal capability. In both groups, long-distance dispersal appears to have played a more significant role in establishing modern patterns than had previously been assumed. Both families also contain examples of recent diversification of species-rich lineages. An understanding of the processes responsible for shaping the distribution patterns of these families has contributed to our understanding of the historical assembly of the biomes that they occupy.

Species delimitation and the origin of populations in island representatives of Phylica (Rhamnaceae).

Relationships between the closely related island species of Phylica (Rhamnaceae) and a mainland species, P. paniculata, were elucidated using amplified fragment length polymorphisms (AFLPs). Parsimony, neighbor joining, and principal coordinate (PCO) analyses indicated that each of the species studied is distinct. AFLPs were also useful in elucidating the genetic relationships and possible infraspecific origins of different island populations in the Atlantic and Indian Oceans. Phylica nitida on Réunion is likely to have been derived from P. nitida on Mauritius. Although the sampling on New Amsterdam is not extensive, the data are also consistent with the hypothesis that P. arborea on New Amsterdam was derived from a single colonization of P. arborea from Gough Island. Similarly, the Gough Island population appears to have been derived from a single colonization event, but it is so distinct from those on Tristan da Cunha, that there may have been two separate dispersals to Gough and Tristan/Nightingale from different lines of the mainland progenitor. There is also evidence of a recolonization from Gough to Tristan da Cunha. Thus, Phylica arborea is capable of repeated long distance dispersal, up to 8000 km, even though the fruits and seeds are not of a type normally associated with this phenomenon.

Population genetic structure of two rare tree species (Colubrina oppositifolia and Alphitonia ponderosa, Rhamnaceae) from Hawaiian dry and mesic forests using random amplified polymorphic DNA markers.

Hawaiian dry and mesic forests contain an increasingly rare assemblage of species due to habitat destruction, invasive alien weeds and exotic pests. Two rare Rhamnaceae species in these ecosystems, Colubrina oppositifolia and Alphitonia ponderosa, were examined using random amplified polymorphic DNA (RAPD) markers to determine the genetic structure of the populations and the amount of variation relative to other native Hawaiian species. Relative variation is lower than with other Hawaiian species, although this is probably not a consequence of genetic bottleneck. Larger populations of both species contain the highest levels of genetic diversity and smaller populations generally the least as determined by number of polymorphic loci, estimated heterozygosity, and Shannon's index of genetic diversity. Populations on separate islands were readily discernible for both species as were two populations of C. oppositifolia on Hawai'i island (North and South Kona populations). Substructure among Kaua'i subpopulations of A. ponderosa that were ecologically separated was also evident. Although population diversity is thought to have remained at predisturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Recovery efforts must focus on control of alien species if these and other endemic dry and mesic forest species are to persist.