Complete Chloroplast Genome of Hypericum perforatum and Dynamic Evolution in Hypericum (Hypericaceae).

Hypericum perforatum (St. John's Wort) is a medicinal plant from the Hypericaceae family. Here, we sequenced the whole chloroplast genome of H. perforatum and compared the genome variation among five Hypericum species to discover dynamic changes and elucidate the mechanisms that lead to genome rearrangements in the Hypericum chloroplast genomes. The H. perforatum chloroplast genome is 139,725 bp, exhibiting a circular quadripartite structure with two copies of inverted repeats (IRs) separating a large single-copy region and a small single-copy region. The H. perforatum chloroplast genome encodes 106 unique genes, including 73 protein-coding genes, 29 tRNAs, and 4 rRNAs. Hypericum chloroplast genomes exhibit genome rearrangement and significant variations among species. The genome size variation among the five Hypericum species was remarkably associated with the expansion or contraction of IR regions and gene losses. Three genes-trnK-UUU, infA, and rps16-were lost, and three genes-rps7, rpl23, and rpl32-were pseudogenized in Hypericum. All the Hypericum chloroplast genomes lost the two introns in clpP, the intron in rps12, and the second intron in ycf3. Hypericum chloroplast genomes contain many long repeat sequences, suggesting a role in facilitating rearrangements. Most genes, according to molecular evolution assessments, are under purifying selection.

Gene loss, genome rearrangement, and accelerated substitution rates in plastid genome of Hypericum ascyron (Hypericaceae).

BACKGROUND: Comparative genomic analysis exhibits dynamic evolution of plastid genome (plastome) in the clusioid clade of Malpighiales, which comprise five families, including multiple inversions and gene losses. Little is known about the plastome evolution in Hypericaceae, a large family in the clade. Only the plastome of one species, Cratoxylum cochinchinense, has been published. RESULTS: We generated a complete plastome sequence for Hypericum ascyron, providing the first complete plastome from the tribe Hypericeae (Hypericaceae). The H. ascyron plastome exhibits dynamic changes in gene and intron content, structure, and sequence divergence compared to the C. cochinchinense plastome from the tribe Cratoxyleae (Hypericaceae). Transcriptome data determined the evolutionary fate of the missing plastid genes infA, rps7, rps16, rpl23, and rpl32 in H. ascyron. Putative functional transfers of infA, rps7, and rpl32 were detected to the nucleus, whereas rps16 and rpl23 were substituted by nuclear-encoded homologs. The plastid rpl32 was integrated into the nuclear-encoded SODcp gene. Our findings suggested that the transferred rpl32 had undergone subfunctionalization by duplication rather than alternative splicing. The H. ascyron plastome rearrangements involved seven inversions, at least three inverted repeat (IR) boundary shifts, which generated gene relocations and duplications. Accelerated substitution rates of plastid genes were observed in the H. ascyron plastome compared with that of C. cochinchinense plastid genes. The higher substitution rates in the accD and clpP were correlated with structural change, including a large insertion of amino acids and losses of two introns, respectively. In addition, we found evidence of positive selection of the clpP, matK, and rps3 genes in the three branches related to H. ascyron. In particular, the matK gene was repeatedly under selection within the family Hypericaceae. Selective pressure in the H. ascyron matK gene was associated with the loss of trnK-UUU and relocation into the IR region. CONCLUSIONS: The Hypericum ascyron plastome sequence provides valuable information for improving the understanding of plastome evolution among the clusioid of the Malpighiales. Evidence for intracellular gene transfer from the plastid to the nucleus was detected in the nuclear transcriptome, providing insight into the evolutionary fate of plastid genes in Hypericaceae.

Calophyllaceae plastomes, their structure and insights in relationships within the clusioids.

A complete chloroplast genome is not yet available for numerous species of plants. Among the groups that lack plastome information is the clusioid clade (Malpighiales), which includes five families: Bonnetiaceae, Calophyllaceae, Clusiaceae, Hypericaceae, and Podostemaceae. With around 2200 species, it has few published plastomes and most of them are from Podostemaceae. Here we assembled and compared six plastomes from members of the clusioids: five from Calophyllaceae (newly sequenced) and one from Clusiaceae. Putative regions for evolutionary studies were identified and the newly assembled chloroplasts were analyzed with other available chloroplasts for the group, focusing on Calophyllaceae. Our results mostly agree with recent studies which found a general conserved structure, except for the two Podostemaceae species that have a large inversion (trnK-UUU-rbcL) and lack one intron from ycf3. Within Calophyllaceae we observed a longer LSC and reduced IRs in Mahurea exstipulata, resulting in some genic rearrangement, and a short inversion (psbJ-psbE) in Kielmeyera coriacea. Phylogenetic analyses recovered the clusioids and the five families as monophyletic and revealed that conflicts in relationships reported in the literature for the group agree with nodes concentrating uninformative or conflicting gene trees. Our study brings new insights about clusioid plastome architecture and its evolution.

Plastome Structural Conservation and Evolution in the Clusioid Clade of Malpighiales.

The clusioid clade of Malpighiales is comprised of five families: Bonnetiaceae, Calophyllaceae, Clusiaceae, Hypericaceae and Podostemaceae. Recent studies have found the plastome structure of Garcinia mangostana L. from Clusiaceae was conserved, while plastomes of five riverweed species from Podostemaceae showed significant structural variations. The diversification pattern of plastome structure of the clusioid clade worth a thorough investigation. Here we determined five complete plastomes representing four families of the clusioid clade. Our results found that the plastomes of the early diverged three families (Clusiaceae, Bonnetiaceae and Calophyllaceae) in the clusioid clade are relatively conserved, while the plastomes of the other two families show significant variations. The Inverted Repeat (IR) regions of Tristicha trifaria and Marathrum foeniculaceum (Podostemaceae) are greatly reduced following the loss of the ycf1 and ycf2 genes. An inversion over 50 kb spanning from trnK-UUU to rbcL in the LSC region is shared by Cratoxylum cochinchinense (Hypericaceae), T. trifaria and Ma. foeniculaceum (Podostemaceae). The large inversed colinear block in Hypericaceae and Podostemaceae contains all the genes in the 50-kb inversed colinear block in a clade of Papilionoideae, with two extra genes (trnK-UUU and matK) at one end. Another endpoint of both inversions in the two clusioids families and Papilionoideae is located between rbcL and accD. This study greatly helped to clarify the plastome evolution in the clusioid clade.

First genomic microsatellite markers developed for Platonia insignis (Clusiaceae), a Brazilian fruit tree.

Platonia insignis is a fruit tree native of Brazil with allogamous and asexual reproduction. The production of fruits is mainly obtained by exploitation of natural populations and the impact of genetic structuring on plant production may be evaluated. For this purpose, codominant and multiallelic markers such as microsatellite are the most suitable, but they need to be developed for this species. Thus, the aim of this work was to develop and validate microsatellite markers for P. insignis. We used Roche 454 GS FLX sequencing platform of a single P. insignis genotype and 1702 microsatellite sequences were identified. Based on some pre-requisites, we could develop 50 primer pairs to be tested. Twenty-two primer pairs successfully amplified fragments and they were tested in 31 genotypes of P. insignis that belong to a germplasm bank and were sampled in the northeast of Pará State, Brazil. Thirteen primers were polymorphic and the number of alleles per loci varied from 5 (PI18 and PI27) to 2 (PI08, PI25, PI31, PI33 and PI 37). Expected heterozygosity (HE) varied from 0.74 (PI27) to 0.12 (PI31) and observed heterozygosity (HO) varied from 1.00 (PI25) to 0.00 (PI08, PI31, PI33 and PI37). Principal coordinates could separate the genotypes of P. insignis in clusters and we can conclude that the primers can estimate the genetic diversity of P. insignis populations.

Plastomes resolve generic limits within tribe Clusieae (Clusiaceae) and reveal the new genus Arawakia.

Clusieae is an exclusively Neotropical tribe in the family Clusiaceae sensu stricto. Although tribes within Clusiaceae are morphologically and phylogenetically well-delimited, resolution among genera within these tribes remains elusive. The tribe Clusieae includes an estimated ∼500 species distributed among five genera: Chrysochlamys, Clusia, Dystovomita, Tovomita, and Tovomitopsis. In this study, we used nearly complete plastid genomes from 30 exemplar Clusieae species representing all genera recognized, plus two outgroups to infer the phylogeny of the tribe using Maximum Likelihood and Bayesian Inference. For comparison, we also inferred a phylogeny from the nuclear Internal Transcribed Spacer (ITS) region using the same methods. Our study corroborates earlier findings that Clusia is monophyletic while Tovomita is not. It also provides additional support to the hypothesis that Chrysochlamys and Tovomitopsis are not closely related despite gross morphological similarity. Tovomita is divided into three distantly related clades: (i) core Tovomita (including the type T. guianensis), (ii) T. croatii, and (iii) the T. weddelliana species complex. Members of the T. weddelliana complex are isolated from the core Tovomita, and placed in a well-supported clade that is sister to a clade composed of Chrysochlamys plus Clusia. Tovomita croatii is nested within Chrysochlamys. We propose taxonomic revisions to accommodate our phylogenetic findings, including the description of the new genus Arawakia, which includes the 18 species formerly recognized in the T. weddelliana species complex. Lectotypes are also designated for nine species (i.e., Arawakia angustata, A. lanceolata, A. lingulata, A. longicuneata, A. macrocarpa, A. oblanceolata, A. pithecobia, A. rhizophoroides, and A. weddelliana), and a taxonomic key for the identification of the six genera of Clusieae recognized is presented.

Altitudinal gradients, biogeographic history and microhabitat adaptation affect fine-scale spatial genetic structure in African and Neotropical populations of an ancient tropical tree species.

The analysis of fine-scale spatial genetic structure (FSGS) within populations can provide insights into eco-evolutionary processes. Restricted dispersal and locally occurring genetic drift are the primary causes for FSGS at equilibrium, as described in the isolation by distance (IBD) model. Beyond IBD expectations, spatial, environmental or historical factors can affect FSGS. We examined FSGS in seven African and Neotropical populations of the late-successional rain forest tree Symphonia globulifera L. f. (Clusiaceae) to discriminate the influence of drift-dispersal vs. landscape/ecological features and historical processes on FSGS. We used spatial principal component analysis and Bayesian clustering to assess spatial genetic heterogeneity at SSRs and examined its association with plastid DNA and habitat features. African populations (from Cameroon and São Tomé) displayed a stronger FSGS than Neotropical populations at both marker types (mean Sp = 0.025 vs. Sp = 0.008 at SSRs) and had a stronger spatial genetic heterogeneity. All three African populations occurred in pronounced altitudinal gradients, possibly restricting animal-mediated seed dispersal. Cyto-nuclear disequilibria in Cameroonian populations also suggested a legacy of biogeographic history to explain these genetic patterns. Conversely, Neotropical populations exhibited a weaker FSGS, which may reflect more efficient wide-ranging seed dispersal by Neotropical bats and other dispersers. The population from French Guiana displayed an association of plastid haplotypes with two morphotypes characterized by differential habitat preferences. Our results highlight the importance of the microenvironment for eco-evolutionary processes within persistent tropical tree populations.

Development of genomic tools in a widespread tropical tree, Symphonia globulifera L.f.: a new low-coverage draft genome, SNP and SSR markers.

Population genetic studies in tropical plants are often challenging because of limited information on taxonomy, phylogenetic relationships and distribution ranges, scarce genomic information and logistic challenges in sampling. We describe a strategy to develop robust and widely applicable genetic markers based on a modest development of genomic resources in the ancient tropical tree species Symphonia globulifera L.f. (Clusiaceae), a keystone species in African and Neotropical rainforests. We provide the first low-coverage (11X) fragmented draft genome sequenced on an individual from Cameroon, covering 1.027 Gbp or 67.5% of the estimated genome size. Annotation of 565 scaffolds (7.57 Mbp) resulted in the prediction of 1046 putative genes (231 of them containing a complete open reading frame) and 1523 exact simple sequence repeats (SSRs, microsatellites). Aligning a published transcriptome of a French Guiana population against this draft genome produced 923 high-quality single nucleotide polymorphisms. We also preselected genic SSRs in silico that were conserved and polymorphic across a wide geographical range, thus reducing marker development tests on rare DNA samples. Of 23 SSRs tested, 19 amplified and 18 were successfully genotyped in four S. globulifera populations from South America (Brazil and French Guiana) and Africa (Cameroon and São Tomé island, FST  = 0.34). Most loci showed only population-specific deviations from Hardy-Weinberg proportions, pointing to local population effects (e.g. null alleles). The described genomic resources are valuable for evolutionary studies in Symphonia and for comparative studies in plants. The methods are especially interesting for widespread tropical or endangered taxa with limited DNA availability.

Characterization saprobic fungi on leaf litter of two species of trees in the Atlantic Forest, Brazil

Abstract We investigated the composition and structure of fungal communities associated with leaf litter generated by Clusia nemorosa and Vismia guianensis that belong to phylogenetically-related botanical families and exist together in a remnant of the Atlantic Forest in Bahia, Brazil. Samplings were conducted during wet (June 2011) and dry (January 2013) seasons in Serra da Jibóia. The fungi were isolated using particle filtration and the 1,832 isolates represented 92 taxa. The wet season yielded the largest number of isolates (1,141) and taxa (76) compared with the dry season (641 isolates and 37 taxa). The richness and diversity of fungal species associated with C. nemorosa (64 taxa, Simpson=0.95)were higher compared with those of V.guianensis (59 taxa, Simpson =0.90). Analysis of similarity (ANOSIM) revealed significant variations in the composition and community structure of fungi isolated from the two plants as a function of seasons. In contrast, nonmetric multidimensional scaling (NMDS) analysis show that the seasonality was an important influence on the distribution of fungal species. However, the populations of the saprobic fungal communities were dynamic, and several factors may influence such communities in the Atlantic Forest.

High-throughput transcriptome sequencing and preliminary functional analysis in four Neotropical tree species.

BACKGROUND: The Amazonian rainforest is predicted to suffer from ongoing environmental changes. Despite the need to evaluate the impact of such changes on tree genetic diversity, we almost entirely lack genomic resources. RESULTS: In this study, we analysed the transcriptome of four tropical tree species (Carapa guianensis, Eperua falcata, Symphonia globulifera and Virola michelii) with contrasting ecological features, belonging to four widespread botanical families (respectively Meliaceae, Fabaceae, Clusiaceae and Myristicaceae). We sequenced cDNA libraries from three organs (leaves, stems, and roots) using 454 pyrosequencing. We have developed an R and bioperl-based bioinformatic procedure for de novo assembly, gene functional annotation and marker discovery. Mismatch identification takes into account single-base quality values as well as the likelihood of false variants as a function of contig depth and number of sequenced chromosomes. Between 17103 (for Symphonia globulifera) and 23390 (for Eperua falcata) contigs were assembled. Organs varied in the numbers of unigenes they apparently express, with higher number in roots. Patterns of gene expression were similar across species, with metabolism of aromatic compounds standing out as an overrepresented gene function. Transcripts corresponding to several gene functions were found to be over- or underrepresented in each organ. We identified between 4434 (for Symphonia globulifera) and 9076 (for Virola surinamensis) well-supported mismatches. The resulting overall mismatch density was comprised between 0.89 (S. globulifera) and 1.05 (V. surinamensis) mismatches/100 bp in variation-containing contigs. CONCLUSION: The relative representation of gene functions in the four transcriptomes suggests that secondary metabolism may be particularly important in tropical trees. The differential representation of transcripts among tissues suggests differential gene expression, which opens the way to functional studies in these non-model, ecologically important species. We found substantial amounts of mismatches in the four species. These newly identified putative variants are a first step towards acquiring much needed genomic resources for tropical tree species.

The ancient tropical rainforest tree Symphonia globulifera L. f. (Clusiaceae) was not restricted to postulated Pleistocene refugia in Atlantic Equatorial Africa.

Understanding the history of forests and their species' demographic responses to past disturbances is important for predicting impacts of future environmental changes. Tropical rainforests of the Guineo-Congolian region in Central Africa are believed to have survived the Pleistocene glacial periods in a few major refugia, essentially centred on mountainous regions close to the Atlantic Ocean. We tested this hypothesis by investigating the phylogeographic structure of a widespread, ancient rainforest tree species, Symphonia globulifera L. f. (Clusiaceae), using plastid DNA sequences (chloroplast DNA [cpDNA], psbA-trnH intergenic spacer) and nuclear microsatellites (simple sequence repeats, SSRs). SSRs identified four gene pools located in Benin, West Cameroon, South Cameroon and Gabon, and São Tomé. This structure was also apparent at cpDNA. Approximate Bayesian Computation detected recent bottlenecks approximately dated to the last glacial maximum in Benin, West Cameroon and São Tomé, and an older bottleneck in South Cameroon and Gabon, suggesting a genetic effect of Pleistocene cycles of forest contraction. CpDNA haplotype distribution indicated wide-ranging long-term persistence of S. globulifera both inside and outside of postulated forest refugia. Pollen flow was four times greater than that of seed in South Cameroon and Gabon, which probably enabled rapid population recovery after bottlenecks. Furthermore, our study suggested ecotypic differentiation-coastal or swamp vs terra firme-in S. globulifera. Comparison with other tree phylogeographic studies in Central Africa highlighted the relevance of species-specific responses to environmental change in forest trees.

Molecular systematic analysis reveals cryptic tertiary diversification of a widespread tropical rain forest tree.

The broad geographic range of many Neotropical rain forest tree species implies excellent dispersal abilities or range establishment that preceded the formation of current dispersal barriers. In order to initiate historical analyses of such widespread Neotropical trees, we sequenced the nuclear ribosomal spacer (ITS) region of Symphonia globulifera L. f. (Clusiaceae) from populations spanning the Neotropics and western Africa. This rain forest tree has left unmistakable Miocene fossils in Mesoamerica (15.5-18.2 Ma) and in South America ( approximately 15 Ma). Although marine dispersal of S. globulifera is considered improbable, our study establishes three marine dispersal events leading to the colonization of Mesoamerica, the Amazon basin, and the West Indies, thus supporting the paleontological data. Our phylogeographic analysis revealed the spatial extent of the three Neotropical S. globulifera clades, which represent trans-Andes (Mesoamerica+west Ecuador), cis-Andes (Amazonia+Guiana), and the West Indies. Strong phylogeographic structure found among trans-Andean populations of S. globulifera stands in contrast to an absence of ITS nucleotide variation across the Amazon basin and indicates profound regional differences in the demographic history of this rain forest tree. Drawing from these results, we provide a historical biogeographic hypothesis to account for differences in the patterns of beta diversity within Mesoamerican and Amazonian forests.