Genomic resources for the Neotropical tree genus Cedrela (Meliaceae) and its relatives.

BACKGROUND: Tree species in the genus Cedrela P. Browne are threatened by timber overexploitation across the Neotropics. Genetic identification of processed timber can be used to supplement wood anatomy to assist in the taxonomic and source validation of protected species and populations of Cedrela. However, few genetic resources exist that enable both species and source identification of Cedrela timber products. We developed several 'omic resources including a leaf transcriptome, organelle genome (cpDNA), and diagnostic single nucleotide polymorphisms (SNPs) that may assist the classification of Cedrela specimens to species and geographic origin and enable future research on this widespread Neotropical tree genus. RESULTS: We designed hybridization capture probes to enrich for thousands of genes from both freshly preserved leaf tissue and from herbarium specimens across eight Meliaceae species. We first assembled a draft de novo transcriptome for C. odorata, and then identified putatively low-copy genes. Hybridization probes for 10,001 transcript models successfully enriched 9795 (98%) of these targets, and analysis of target capture efficiency showed that probes worked effectively for five Cedrela species, with each species showing similar mean on-target sequence yield and depth. The probes showed greater enrichment efficiency for Cedrela species relative to the other three distantly related Meliaceae species. We provide a set of candidate SNPs for species identification of four of the Cedrela species included in this analysis, and present draft chloroplast genomes for multiple individuals of eight species from four genera in the Meliaceae. CONCLUSIONS: Deforestation and illegal logging threaten forest biodiversity globally, and wood screening tools offer enforcement agencies new approaches to identify illegally harvested timber. The genomic resources described here provide the foundation required to develop genetic screening methods for Cedrela species identification and source validation. Due to their transferability across the genus and family as well as demonstrated applicability for both fresh leaves and herbarium specimens, the genomic resources described here provide additional tools for studies examining the ecology and evolutionary history of Cedrela and related species in the Meliaceae.

Phytochemistry and pharmacology of the genus Entandrophragma over the 50 years from 1967 to 2018: a 'golden' overview.

OBJECTIVES: For centuries, the genus Entandrophragma (Meliaceae), endemic to Africa, has been used in traditional medicine for the treatment of several illnesses. This review deals with large range of phytochemicals from the genus Entandrophragma and their pharmacological potentials covering the period from 1967 to 2018. KEY FINDINGS: Phytochemical investigations of the genus Entandrophragma led to the report of about 166 secondary metabolites which have been thoroughly summarized herein including some of their semisynthetic derivatives obtained from chemical transformations as well as their biological activities in the medicinal and agricultural domains. The limonoids or meliacins and their precursor called protolimonoids (protomeliacins) reported so far represent almost 69.28% of the total secondary metabolites obtained from the genus, and they display the most potent biological activities. Collectively, both classes of metabolites constitute the markers of Entandrophragma. However, squalene-type triterpenoids and sesquiterpenoids were reported only from the species E. cylindricum and therefore could be its chemotaxonomic markers. SUMMARY: The pharmacological investigations of the extracts of some species exhibited interesting results which support the traditional uses of these Entandrophragma plants in folk medicine. Some compounds revealed promising antiplasmodial and anti-inflammatory activities and deserve therefore further attention for new drug discovery.

Complete Chloroplast Genome Sequences of Four Meliaceae Species and Comparative Analyses.

The Meliaceae family mainly consists of trees and shrubs with a pantropical distribution. In this study, the complete chloroplast genomes of four Meliaceae species were sequenced and compared with each other and with the previously published Azadirachta indica plastome. The five plastomes are circular and exhibit a quadripartite structure with high conservation of gene content and order. They include 130 genes encoding 85 proteins, 37 tRNAs and 8 rRNAs. Inverted repeat expansion resulted in a duplication of rps19 in the five Meliaceae species, which is consistent with that in many other Sapindales, but different from many other rosids. Compared to Azadirachta indica, the four newly sequenced Meliaceae individuals share several large deletions, which mainly contribute to the decreased genome sizes. A whole-plastome phylogeny supports previous findings that the four species form a monophyletic sister clade to Azadirachta indica within the Meliaceae. SNPs and indels identified in all complete Meliaceae plastomes might be suitable targets for the future development of genetic markers at different taxonomic levels. The extended analysis of SNPs in the matK gene led to the identification of four potential Meliaceae-specific SNPs as a basis for future validation and marker development.

Genetic diversity of central and peripheral populations of Toona ciliata var. pubescens, an endangered tree species endemic to China.

Our objective was to examine the genetic diversity of central and peripheral populations of Toona ciliata var. pubescens, to elucidate whether the central-peripheral hypothesis applies to these populations. We analyzed 392 individuals from nine natural populations using eight pairs of polymorphic SSR primers. The results showed that the mean numbers of observed and expected alleles in peripheral populations were higher than in central populations. Common widespread and rare local (RL) alleles were observed in all populations. However, common local alleles were found in five populations and rare widespread alleles were only found in three. The total numbers of the four types of alleles were higher in peripheral than in central populations, and the quantity of the RL allele was obviously higher in the peripheral populations than in the central populations. Both the observed and expected heterozygosities were higher in peripheral populations compared with the central populations. The coefficient of gene differentiation of the peripheral populations was 0.3045, which was significantly higher than that of the central populations. The gene flow between central populations was greater than one, but less than one between peripheral populations. This indicates that frequent gene flow exists between central populations, while terrain and habitat fragmentation prevent gene flow between peripheral populations. A Mantel test indicated that there was no relationship between genetic and geographical distance of T. ciliata var. pubescens.

An evaluation of taxonomic concepts of the widespread plant genus Aglaia and its allies across Wallace's Line (tribe Aglaieae, Meliaceae).

Similar to other species-rich taxa in the Indo-Australian Archipelago, taxonomy of the genus Aglaia (mahogany family, Meliaceae) remains problematic. This study aims to evaluate taxonomic concepts within Aglaia based on the largest dataset to-date. We analyzed sequences of 237 accessions of Aglaia and representatives of all other genera of the tribe Aglaieae, including nuclear ribosomal ITS, the trnL-trnF intron and intergenic spacer, the atpF intron and the petD region comprising the petB-petD spacer, the petD-5' exon and the petD intron (all but the first from the plastid genome). Our analyses were set both in maximum likelihood and Bayesian frameworks, which (1) supported paraphyly of Aglaia and Aphanamixis; (2) demonstrated polyphyly of previously described sections for Aglaia; and (3) suggested delimitation problems with 57% of the morphologically "variable species" and all "complex species". In general, there were more genetic entities than species described, which shows that the taxonomy of this group is more complex than has sometimes been previously assumed. For some species, morphological variation suggests the existence of more variants, subspecies or species within various taxa. Furthermore, our study detected additional phylogenetic entities that were geographically distinct, occurring on either side of Wallace's Line but not on both sides. The delineation of these inter-specific taxa needs further investigation by taking into account the morphological variation within and between populations across the entire distribution.

Quantificação de fenóis totais presentes nas cascas de Trichillia catigua A. Juss (Meliaceae) / Quantification of total phenols present in the bark of Trichilia catigua A. Juss (Meliaceae)

Trichillia catigua A. Juss. é uma planta medicinal utilizada principalmente como estimulante, possivelmente devido à presença de fenóis. Entre os métodos utilizados para a determinação de polifenóis em matérias-primas vegetais destaca-se a análise por meio de ensaios espectrofotométricos por apresentarem boa sensibilidade, simplicidade, e ter custos mais acessíveis. O presente trabalho teve o objetivo de validar a metodologia analítica para determinação de polifenóis na casca de catuaba em pó. Utilizou-se o método de Hagerman & Butler para a quantificação dos polifenóis totais encontrando-se 7,96%. O método validado mostrou-se simples, rápido, seletivo, linear, exato, preciso, e robusto para ser executado na rotina de laboratório de controle de qualidade, constituindo uma possibilidade de análise desta planta medicinal.

Trichilia catigua A. Juss. is a medicinal plant used especially as a stimulant, possibly due to the presence of phenols. Among the methods used for the determination of polyphenols in raw plant materials is analysis through spectrophotometric assays since they have good sensitivity, are simple and more affordable. This study aimed to validate the analytical methodology for the determination of polyphenols in the bark of powder "catuaba". The method of Hagerman & Butler was employed in the quantification of total polyphenols, yielding 7.96%. The validated method has proven to be simple, rapid, selective, linear, accurate, precise and robust to be performed in the routine of a laboratory of quality control, constituting a possibility for the analysis of this medicinal plant.

Rapid microsatellite marker development for African mahogany (Khaya senegalensis, Meliaceae) using next-generation sequencing and assessment of its intra-specific genetic diversity.

Khaya senegalensis (African mahogany or dry-zone mahogany) is a high-value hardwood timber species with great potential for forest plantations in northern Australia. The species is distributed across the sub-Saharan belt from Senegal to Sudan and Uganda. Because of heavy exploitation and constraints on natural regeneration and sustainable planting, it is now classified as a vulnerable species. Here, we describe the development of microsatellite markers for K. senegalensis using next-generation sequencing to assess its intra-specific diversity across its natural range, which is a key for successful breeding programs and effective conservation management of the species. Next-generation sequencing yielded 93,943 sequences with an average read length of 234 bp. The assembled sequences contained 1030 simple sequence repeats, with primers designed for 522 microsatellite loci. Twenty-one microsatellite loci were tested with 11 showing reliable amplification and polymorphism in K. senegalensis. The 11 novel microsatellites, together with one previously published, were used to assess 73 accessions belonging to the Australian K. senegalensis domestication program, sampled from across the natural range of the species. STRUCTURE analysis shows two major clusters, one comprising mainly accessions from west Africa (Senegal to Benin) and the second based in the far eastern limits of the range in Sudan and Uganda. Higher levels of genetic diversity were found in material from western Africa. This suggests that new seed collections from this region may yield more diverse genotypes than those originating from Sudan and Uganda in eastern Africa.

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae).

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.

Evaluation of candidate DNA barcoding loci for economically important timber species of the mahogany family (Meliaceae).

There has been considerable debate regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from proposed candidate loci on a common set of samples. In this study, we evaluated main candidate plastid regions (rpoC1, rpoB, accD) and additional plastid markers (psbB, psbN, psbT exons and the trnS-trnG spacer) as well as the nuclear ribosomal spacer region (ITS1-5.8S-ITS2) in a group of land plants belonging to the mahogany family, Meliaceae. Across these samples, only ITS showed high levels of resolvability. Interspecific sharing of sequences from individual plastid loci was common. The combination of multiple loci did not improve performance. DNA barcoding with ITS alone revealed cryptic species and proved useful in identifying species listed in Convention on International Trade of Endangered Species appendixes.

An overview of the genus Xylocarpus.

The article deals with the chemical constituents isolated from all the Indian species of Xylocarpus viz. X. granatum, X. moluccensis, and X. mekongensis. A total of about 70 pure compounds of different classes have been covered. The article describes the biosynthetic pathway of limonoids and sources, bioactivities and chemical and physical constants of the different classes of compounds.

Classification of commercial Catuaba samples by NMR, HPLC and chemometrics.

For over a century, Catuaba has been used in Brazilian folk medicine as an aphrodisiac even though the identity of the plant material employed is often uncertain. The species recommended by the Brazilian Pharmacopeia is Anemopaegma arvense (Bignoniaceae), but many other plants, regionally known as Catuaba, are commercialised. Frequently, the quality control of such a complex system is based on chemical markers that do not supply a general idea of the system. With the advent of the metabolomics approach, a global analysis of samples becomes possible. It appears that (1)H-NMR is the most useful method for such application, since it can be used as a wide-spectrum chemical analysis technique. Unfortunately, the generated spectra is complex so a possible approach is to look at the metabolite profile as a whole using multivariate methods, for example, by application of principal component analysis (PCA). In the present paper, we describe for the first time a proton high-resolution magic angle spinning nuclear magnetic resonance ((1)H-HR-MAS NMR) method coupled with PCA for the metabolomic analysis of some commercial Catuaba samples, which provided a reduction in the time required for such analysis. A comparative study of HPLC, HR-MAS and liquid-NMR techniques is also reported.

Blind population genetics survey of tropical rainforest trees.

Rainforest tree species can be difficult to identify outside of their period of reproduction. Vascular tissues from Carapa spp. individuals were collected during a short field trip in French Guiana and analysed in the laboratory with nuclear and chloroplast markers. Using a Bayesian approach, > 90% of the samples could be assigned to one of two distinct clusters corresponding to previously described species, making it possible to estimate the genetic structure of each species and to identify cases of introgression. We argue that this blind procedure represents a first-choice rather than a fallback option whenever related taxa are investigated.

Application of high-performance liquid chromatography-nuclear magnetic resonance coupling to the identification of limonoids from mahogany tree (Switenia macrophylla, Meliaceae) by stopped-flow 1D and 2D NMR spectroscopy.

Separation and characterization of limonoids from Switenia macrophylla (Meliaceae) by HPLC-NMR technique has been described. Analyses were carried out using reversed-phase gradient HPLC elution coupled to NMR (600 MHz) spectrometer in stopped-flow mode. Separated peaks were collected into an interface unit prior to NMR measurements, which were performed with suppression of solvent signals by shaped pulses sequences. Structure elucidation of the limonoids was attained by data obtained from 1H NMR, TOCSY, gHSQC and gHMBC spectra without conventional isolation that is usually applied in natural products studies.

The mahogany family "out-of-Africa": divergence time estimation, global biogeographic patterns inferred from plastid rbcL DNA sequences, extant, and fossil distribution of diversity.

With information on fossils and extant distribution of diversity/endemism in the mahogany family, we perform a global biogeographic study of Meliaceae using plastid rbcL data for all subfamilies, tribes and nearly all genera. Our study indicates that: (1) Meliaceae are of western Gondwanan origin; (2) dispersal played an important role for the current distribution of mahogany biota; and (3) the direction of dispersal was most likely an "out-of-Africa" scenario with important dispersal routes across Eurasia and between Eurasia and North America provided by Beringia and the North Atlantic land bridge and North America and South America via island chains and/or direct land connections. Populations in North America, Europe, and East Asia were presumably eliminated as tropical climates disappeared from these areas during the Miocene. Extensive Meliaceae fossil findings confirm that the entry of megathermal (frost-intolerant) angiosperms into southern continents from Oligocene to Pliocene must be considered as an important means of establishing pantropical distribution patterns.

Arbuscular mycorrhizal fungi associated with the Meliaceae on Hainan island, China.

Species richness, spore density, frequency of occurrence, and relative abundance of AM fungi were determined in rhizosphere soil samples from nine tropical rainforest sites on Hainan island, south China, and the arbuscular mycorrhizal (AM) status of members of the Meliaceae was examined. All 28 plant taxa investigated (25 species including two varieties of 1 species and three varieties of another) were colonized by AM fungi. The mean proportion of root length colonized was 56% (range 10-95%). Vesicles were observed in 27 and hyphal coils in 26 of the 28 plant taxa. Mycorrhizas were of the Paris-type or intermediate-type, with no Arum-type mycorrhizas observed. Species richness of AM fungi varied from 3 to 15 and spore density from 46 to 1,499 per 100 g rhizosphere soil. Of 33 AM fungal taxa in five genera isolated and identified, 18 belonged to Glomus, 9 to Acaulospora, 1 to Entrophospora, 2 to Gigaspora, and 3 to Scutellospora. Acaulospora and Glomus were the dominant genera identified. Glomus claroideum was the taxon most commonly isolated, with a frequency of occurrence of 56.5% and relative abundance of 10.4%. A positive correlation was found between percentage of root length colonization and species richness. However, there was no correlation between spore density and percentage of root length colonized by AM fungi.

Trypanocidal activity of Meliaceae and Rutaceae plant extracts.

The in vitro trypanocidal activity of 22 extracts and 43 fractions of plants belonging to the families Meliaceae and Rutaceae was evaluated. The extracts from leaves of Conchocarphus heterophyllus and branches of Trichilia ramalhoi were the most active. The trypanocidal activity seems to be increased by fractionation of the extracts. Fractions from C. heterophyllus and Galipea carinata were the most active and a 100% lysis of the parasites was observed for five fractions. From one of them were isolated two flavonoids: flavone and 7-methoxyflavone, which showed weak trypanocidal activity. The results obtained from the extracts and fractions revealed that the order Rutales is a promising source for the search of new drugs for Chagas disease. Phytochemical studies with the other active fractions are underway in order to isolate compounds, which could be associated with observed activities.