The complete organellar genomes of the entheogenic plant Psychotria viridis (Rubiaceae), a main component of the ayahuasca brew.

Psychotria viridis (Rubioideae: Rubiaceae), popularly known as chacrona, is commonly found as a shrub in the Amazon region and is well-known to produce psychoactive compounds, such as the N,N-dimethyltryptamine (DMT). Together with the liana Banisteropsis caapi, P. viridis is one of the main components of the Amerindian traditional, entheogenic beverage known as ayahuasca. In this work, we assembled and annotated the organellar genomes (ptDNA and mtDNA), presenting the first genomics resources for this species. The P. viridis ptDNA exhibits 154,106 bp, encoding all known ptDNA gene repertoire found in angiosperms. The Psychotria genus is a complex paraphyletic group, and according to phylogenomic analyses, P. viridis is nested in the Psychotrieae clade. Comparative ptDNA analyses indicate that most Rubiaceae plastomes present conserved ptDNA structures, often showing slight differences at the junction sites of the major four regions (LSC-IR-SSC). For the mitochondrion, assembly graph-based analysis supports a complex mtDNA organization, presenting at least two alternative and circular mitogenomes structures exhibiting two main repeats spanning 24 kb and 749 bp that may symmetrically isomerize the mitogenome into variable arrangements and isoforms. The circular mtDNA sequences (615,370 and 570,344 bp) encode almost all plant mitochondrial genes (except for the ccmC, rps7, rps10, rps14, rps19, rpl2 and rpl16 that appears as pseudogenes, and the absent genes sdh3, rps2, rsp4, rsp8, rps11, rpl6, and rpl10), showing slight variations related to exclusive regions, ptDNA integration, and relics of previous events of LTR-RT integration. The detection of two mitogenomes haplotypes is evidence of heteroplasmy as observed by the complex organization of the mitochondrial genome using graph-based analysis. Taken together, these results elicit the primary insights into the genome biology and evolutionary history of Psychotria viridis and may be used to aid strategies for conservation of this sacred, entheogenic species.

Reproductive success and genetic diversity of Psychotria hastisepala (Rubiaceae), in fragmented Atlantic forest, Southeastearn Brazil.

The impacts of forest fragmentation on both reproductive biology and genetic diversity of native plant species is hardly understood, despite some studies have analyzed this current worldwide problem. Since this constitutes one of the main threats to seasonal semi-deciduous forests in Southeastern Brazil, we investigated the reproductive success and the genetic diversity of a distylous, understory shrub (Psychotria hastisepala) within this context of forest fragmentation. For this study, a set of seven forest fragments of sizes ranging from 4.1 to 168.7 hectares were chosen. The intervenient matrix comprised pastures (25-50%), monocultures (33-50%) and rural roads and buildings (14-28.5%). Overall, 91 plants (54 for the short-styled morph and 37 for the long-styled morph; mean of 6.5 plants per fragment) were investigated. To evaluate reproductive success, we quantified fruit and seed production under natural pollination; to evaluate genetic diversity and population structure, we employed ISSR markers on genomic DNA. Plants with the short-styled morph exhibited a significantly higher reproductive success than those with the long-styled morph; there was no association between seed production and size of the forest fragment. Levels of genetic diversity were positively associated with the number of plants per fragment; but they were not related to flower morph. AMOVA showed that about 65% of the overall genetic variation was attributed to the differences between plants within fragments. The results suggested that populations of P. hastisepala were susceptible to decline owing to forest fragmentation.

Reproductive success and genetic diversity of Psychotria hastisepala (Rubiaceae), in fragmented Atlantic forest, Southeastearn Brazil

The impacts of forest fragmentation on both reproductive biology and genetic diversity of native plant species is hardly understood, despite some studies have analyzed this current worldwide problem. Since this constitutes one of the main threats to seasonal semi-deciduous forests in Southeastern Brazil, we investigated the reproductive success and the genetic diversity of a distylous, understory shrub (Psychotria hastisepala) within this context of forest fragmentation. For this study, a set of seven forest fragments of sizes ranging from 4.1 to 168.7 hectares were chosen. The intervenient matrix comprised pastures (25-50%), monocultures (33-50%) and rural roads and buildings (14-28.5%). Overall, 91 plants (54 for the short-styled morph and 37 for the long-styled morph; mean of 6.5 plants per fragment) were investigated. To evaluate reproductive success, we quantified fruit and seed production under natural pollination; to evaluate genetic diversity and population structure, we employed ISSR markers on genomic DNA. Plants with the short-styled morph exhibited a significantly higher reproductive success than those with the long-styled morph; there was no association between seed production and size of the forest fragment. Levels of genetic diversity were positively associated with the number of plants per fragment; but they were not related to flower morph. AMOVA showed that about 65% of the overall genetic variation was attributed to the differences between plants within fragments. The results suggested that populations of P. hastisepala were susceptible to decline owing to forest fragmentation. Rev. Biol. Trop. 62 (1): 309-319. Epub 2014 March 01.

Los impactos de la fragmentación de los bosques tanto en la biología reproductiva como en la diversidad genética de las especies de plantas nativas apenas se entienden, a pesar de que algunos estudios han analizado este problema mundial. Dado que esto constituye una de las principales amenazas para los bosques semi-deciduos estacionales en el sureste de Brasil, se investigó el éxito reproductivo y la diversidad genética de un arbusto de sotobosque, Psychotria hastisepala, en un bosque fragmentado. Para este estudio, se seleccionaron siete fragmentos de tamaños que van desde 4.1 hasta 168.7 hectáreas. La matriz intervenida comprende pastos (25-50%), monocultivos (33-50%) y carreteras y edificios rurales (14-28.5%). En total, 91 plantas fueron investigadas (54 morfos florales brevistilo y 37 longistilo, con una media de 6.5 plantas por fragmento). Para evaluar el éxito reproductivo, se cuantificó la producción de frutos y semillas de plantas con polinización natural; para evaluar la diversidad genética y la estructura poblacional, se emplearon marcadores ISSR en el ADN genómico. Las plantas con morfo brevistilo exhibieron un éxito reproductivo significativamente más alto que las longistilo, no hubo asociación entre la producción de semillas y el tamaño del fragmento de bosque. Los niveles de diversidad genética se asociaron positivamente con el número de plantas por fragmento, pero que no estaban relacionados con los morfos florales. AMOVA mostró que alrededor del 65% de la variación genética en general se atribuyó a las diferencias entre plantas dentro de fragmentos. Los resultados sugieren que las poblaciones de P. hastisepala fueron susceptibles a disminuir debido a la fragmentación de los bosques.

The metal transporter PgIREG1 from the hyperaccumulator Psychotria gabriellae is a candidate gene for nickel tolerance and accumulation.

Nickel is an economically important metal and phytotechnologies are being developed to limit the impact of nickel mining on the environment. More than 300 plant species are known to hyperaccumulate nickel. However, our knowledge of the mechanisms involved in nickel accumulation in plants is very limited because it has not yet been possible to study these hyperaccumulators at the genomic level. Here, we used next-generation sequencing technologies to sequence the transcriptome of the nickel hyperaccumulator Psychotria gabriellae of the Rubiaceae family, and used yeast and Arabidopsis as heterologous systems to study the activity of identified metal transporters. We characterized the activity of three metal transporters from the NRAMP and IREG/FPN families. In particular, we showed that PgIREG1 is able to confer nickel tolerance when expressed in yeast and in transgenic plants, where it localizes in the tonoplast. In addition, PgIREG1 shows higher expression in P. gabriellae than in the related non-accumulator species Psychotria semperflorens. Our results designate PgIREG1 as a candidate gene for nickel tolerance and hyperaccumulation in P. gabriellae. These results also show how next-generation sequencing technologies can be used to access the transcriptome of non-model nickel hyperaccumulators to identify the underlying molecular mechanisms.

New Caledonian lineages of Psychotria (Rubiaceae) reveal different evolutionary histories and the largest documented plant radiation for the archipelago.

New Caledonia is a remote archipelago of the South-West Pacific, whose flora is rich, distinctive, and disharmonic. The interest of botanists has long been attracted by the spatio-temporal origin of this flora, but little attention has been paid to the modes of colonization and the diversification processes that have led to the archipelago's modern flora. To date, no explosive plant radiation has yet been highlighted for New Caledonia. A dated phylogenetic framework on the second richest New Caledonian genus--Psychotria s.l. and its allied genera (tribes Psychotrieae and Palicoureeae, Rubiaceae; ca. 85 species)--is provided in this study to explore its patterns of colonization and diversification in the archipelago. This study is based on a comprehensive species sampling, two nuclear and four plastid loci. Results show that New Caledonia was colonized four times by Psychotria and its allied genera during the Neogene long after its mid-Eocene re-emergence from the sea. The Pacific clade of Psychotrieae, one of the largest plant diversifications in the Pacific islands and the Indo-Pacific region, is absent from New Caledonia, possibly due to niche competition. Although the four lineages colonized New Caledonia relatively simultaneously during the Neogene, they express different evolutionary histories, as revealed by unevenness in species richness and net diversification rates. The genus Geophila has not diversified on New Caledonia, as a non-endemic single species has been documented in the archipelago. The genus Margaritopsis had a moderate level of diversification (four species) similar to that on other Pacific islands. The Psychotria clade NC1 appears to be a relictual lineage, which probably underwent a drastic extinction, with a narrow ecological habitat and dispersal limitations. The Psychotria clade NC2 is the largest and youngest New Caledonian plant radiation, and has undergone the fastest recorded diversification of any endemic lineage in the archipelago, and could be the result of a 'non-adaptive radiation', originating from Australian rainforests.

Fine-scale niche structure of Neotropical forests reflects a legacy of the Great American Biotic Interchange.

The tendency of species to retain their ancestral niches may link processes that determine community assembly with biogeographic histories that span geological time scales. Biogeographic history is likely to have had a particularly strong impact on Neotropical forests because of the influence of the Great American Biotic Interchange, which followed emergence of a land connection between North and South America ~3 Ma. Here we examine the community structure, ancestral niches and ancestral distributions of the related, hyperdiverse woody plant genera Psychotria and Palicourea (Rubiaceae) in Panama. We find that 49% of the variation in hydraulic traits, a strong determinant of community structure, is explained by species' origins in climatically distinct biogeographic regions. Niche evolution models for a regional sample of 152 species indicate that ancestral climatic niches are associated with species' habitat distributions, and hence local community structure and composition, even millions of years after dispersal into new geographic regions.

Proteomics analysis of Psychotria leaf nodule symbiosis: improved genome annotation and metabolic predictions.

Several plant species of the genus Psychotria (Rubiaceae) harbor Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted vertically between plant generations and have not yet been cultured outside of their host. This symbiosis is considered to be obligatory because plants devoid of symbionts fail to develop into mature individuals. The genome of 'Candidatus Burkholderia kirkii' has been sequenced recently and has revealed evidence of reductive genome evolution, as shown by the proliferation of insertion sequences and the presence of numerous pseudogenes. We employed shotgun proteomics to investigate the expression of 'Ca. B. kirkii' proteins in the leaf nodule. Drawing from this dataset and refined comparative genomics analyses, we designed a new pseudogene prediction algorithm and improved the genome annotation. We also found conclusive evidence that nodule bacteria allocate vast resources to synthesis of secondary metabolites, possibly of the C7N aminocyclitol family. Expression of a putative 2-epi-5-valiolone synthase, a key enzyme of the C7N aminocyclitol synthesis, is high in the nodule population but downregulated in bacteria residing in the shoot apex, suggesting that production of secondary metabolites is particularly important in the leaf nodule.

Endosymbiont transmission mode in bacterial leaf nodulation as revealed by a population genetic study of Psychotria leptophylla.

Leaf-nodulated plants are colonized by vertically inherited bacterial endosymbionts, which maintain symbioses throughout host generations. The permanent character of the interaction implies phylogenetic congruence between the host and the endosymbiont. However, the present population genetic study of Psychotria leptophylla provides evidence for a mixed symbiont transmission involving both vertical inheritance and horizontal transfers from the environment.

Genetic structure of tree and shrubby species among anthropogenic edges, natural edges, and interior of an atlantic forest fragment.

Two species, Psychotria tenuinervis (shrub, Rubiaceae) and Guarea guidonia (tree, Meliaceae), were used as models to compare the genetic structure of tree and shrubby species among natural edges, anthropogenic edges, and a fragment interior. There were significant differences between two genetic markers. For isozymes, P. tenuinervis presented greater heterozygosity (expected and observed) and a higher percentage of polymorphic loci and median number of alleles than G. guidonia. For microsatellites, there was no difference in genetic variability between the species. Only P. tenuinervis, for isozymes, showed differences in genetic variability among the three habitats. There was no genetic structure (F (ST) < 0.05) among habitats in both plant species for both genetic markers. Isozymes showed great endogamy for both plant species, but not microsatellites. The forest fragmentation may have negative effects on both spatial (among edges and interior) and temporal genetic variability.

Evolutionary time for dispersal limits the extent but not the occupancy of species' potential ranges in the tropical plant genus Psychotria (Rubiaceae).

Explaining the diversity in geographic range sizes among species is a central goal of ecological and evolutionary studies. We tested species age as an explanation of range size variation within a group of understory shrubs in the Neotropics (Psychotria subgenus Psychotria, Rubiaceae). We distinguish between range occupancy (filling an occupied area) and range extent (maximum distances dispersed). We used Bayesian relaxed-clock dating of molecular sequence data to estimate the relative age of species, and we used species distribution modeling to predict species' potential ranges. If the range sizes of species are limited by time for dispersal, we hypothesize that older species should have (1) larger realized range occupancies and realized range extents than younger species, (2) filled a greater proportion of their potential range occupancies, and (3) colonized a greater proportion of their potential range extents. We found (1) a significant but weak positive relationship between species age versus both realized range occupancy and realized range extent, (2) no relationship between species age and filling of potential range occupancies, but (3) that older species had colonized a significantly greater proportion of their potential range extents than younger species. Our results indicate that a time-for-dispersal effect can limit the extent of ranges of species but not necessarily their occupancies.

Mating systems of Psychotria tenuinervis (Rubiaceae): distance from anthropogenic and natural edges of Atlantic forest fragment.

The aim of this study was to determine and compare the mating systems among Psychotria tenuinervis populations at anthropogenic edges, natural edges, and the forest interior using allozyme electrophoresis of naturally pollinated progeny arrays. P. tenuinervis showed low outcrossing rates, varying from 37% to 50% of the mating attributable to outcrossing and 50% to 63% attributable to self-fertilization, in the three habitats. The forest interior had the highest outcrossing rate (t(m) = 0.50 and t(s) = 0.43) among the three habitats. However, there were no differences in either multilocus or single-locus rates among the three habitats, indicating that the contribution of biparental inbreeding to the apparent selfing rate in these populations was very low. The multilocus (t(m)) and single-locus (t(s)) outcrossing rates for the P. tenuinervis in the sample plots within each habitat showed great heterogeneity. In conclusion, edge creation seems not to influence its mating systems. Additionally, although P. tenuinervis is a distylous species, the population's inbreeding can be attributed almost entirely to self-fertilization.

Breeding system and floral morphometry of distylous Psychotria L. species in the Atlantic rain forest, SE Brazil.

General patterns of floral morphology and incompatibility mechanisms have been described for many distylous plants. The absence of these patterns in typically distylous groups, as observed especially in tropical environments, is interpreted as atypical distyly, or as a new reproductive strategy derived from it. Data are presented here on the morphological and compatibility relations between floral morphs of four Psychotria dimorphic species in the Atlantic rain forest in SE Brazil: Psychotria jasminoides, P. birotula, P. mapourioides, and P. pubigera. When significant differences were found, floral parts were larger in thrum flowers. Results of controlled crosses showed that most incompatible pollen tubes were arrested in the stigma, and only in a low proportion in the upper parts of the style. We conclude that, at the study site, the majority and most important morphological and mating features of typical distyly seem to be conserved in P. jasminoides and P. mapourioides, which presented reciprocal herkogamy, self and intramorph incompatibility, and a balanced morph ratio in the population. Typical distyly in P. birotula is supported by floral morphology, pollen tube data and morph ratio and, in P. pubigera, only by floral morphology and pollen tube data.

Reconstructing ancestral patterns of colonization and dispersal in the Hawaiian understory tree genus Psychotria (Rubiaceae): a comparison of parsimony and likelihood approaches.

Systematic and biogeographical relationships within the Hawaiian clade of the pantropical understory shrub genus Psychotria (Rubiaceae) were investigated using phylogenetic analysis of 18S-26S ribosomal DNA internal (ITS) and external (ETS) transcribed spacers. Phylogenetic analyses strongly suggest that the Hawaiian Psychotria are monophyletic and the result of a single introduction to the Hawaiian Islands. The results of phylogenetic analyses of ITS and ETS partitions alone give slightly different topologies among basal lineages of the Hawaiian clade; however, such differences are not well supported. Relationships in the section Straussia clade in particular are not well resolved because of few nucleotide changes on internal branches, suggesting extremely rapid radiation in the lineage. Parsimony and likelihood reconstructions of ancestral geographical distributions using the topologies inferred from both parsimony and likelihood analysis of combined data and using different combinations of models and branch lengths gave highly congruent results. However, for one internal node (corresponding to the majority of the "greenwelliae" clade), parsimony reconstructions were unable to distinguish between three possible island states, whereas likelihood reconstructions resulted in clear ordering of possible states, with the island of Oàhu slightly more probable than other islands under all but one model and branch length combination considered (the Jukes-Cantor-like model with branch lengths inferred under parsimony, under which conditions Maui Nui is more probable). A pattern of colonization from oldest to youngest islands was inferred from the phylogeny, using maximum parsimony and maximum likelihood. Additionally, a much higher incidence of intraisland versus interisland speciation was inferred.