Genomic decoding of Theobroma grandiflorum (cupuassu) at chromosomal scale: evolutionary insights for horticultural innovation.

BACKGROUND: Theobroma grandiflorum (Malvaceae), known as cupuassu, is a tree indigenous to the Amazon basin, valued for its large fruits and seed pulp, contributing notably to the Amazonian bioeconomy. The seed pulp is utilized in desserts and beverages, and its seed butter is used in cosmetics. Here, we present the sequenced telomere-to-telomere genome of cupuassu, disclosing its genomic structure, evolutionary features, and phylogenetic relationships within the Malvaceae family. FINDINGS: The cupuassu genome spans 423 Mb, encodes 31,381 genes distributed in 10 chromosomes, and exhibits approximately 65% gene synteny with the Theobroma cacao genome, reflecting a conserved evolutionary history, albeit punctuated with unique genomic variations. The main changes are pronounced by bursts of long-terminal repeat retrotransposons at postspecies divergence, retrocopied and singleton genes, and gene families displaying distinctive patterns of expansion and contraction. Furthermore, positively selected genes are evident, particularly among retained and dispersed tandem and proximal duplicated genes associated with general fruit and seed traits and defense mechanisms, supporting the hypothesis of potential episodes of subfunctionalization and neofunctionalization following duplication, as well as impact from distinct domestication process. These genomic variations may underpin the differences observed in fruit and seed morphology, ripening, and disease resistance between cupuassu and the other Malvaceae species. CONCLUSIONS: The cupuassu genome offers a foundational resource for both breeding improvement and conservation biology, yielding insights into the evolution and diversity within the genus Theobroma.

DNA Barcodes of Mansonia (Mansonia) Blanchard, 1901 (Diptera, Culicidae).

Females of the genus Mansonia feed on the blood of humans, livestock, and other vertebrates to develop their eggs. The females' biting behavior may cause severe disturbance to blood hosts, with a negative impact on public health and economics. Certain species have been identified as potential or effective disease vectors. The accurate species identification of field-collected specimens is of paramount importance for the success of monitoring and control strategies. Mansonia (Mansonia) morphological species boundaries are blurred by patterns of intraspecific heteromorphism and interspecific isomorphism. DNA barcodes can help to solve taxonomic controversies, especially if combined with other molecular tools. We used cytochrome c oxidase subunit I (COI) gene 5' end (DNA barcode) sequences to identify 327 field-collected specimens of Mansonia (Mansonia) spp. The sampling encompassed males and females collected from three Brazilian regions and previously assigned to species based on their morphological characteristics. Eleven GenBank and BOLD sequences were added to the DNA barcode analyses. Initial morphospecies assignments were mostly corroborated by the results of five clustering methods based on Kimura two-parameter distance and maximum likelihood phylogeny. Five to eight molecular operational taxonomic units may represent taxonomically unknown species. The first DNA barcode records for Mansonia fonsecai, Mansonia iguassuensis, and Mansonia pseudotitillans are presented.

Stomatal size, ploidy and polyembryony in Eriotheca Stellate Trichome Species Complex (Bombacoideae - Malvaceae) in the Cerrados of Brazil.

Geographic parthenogenesis, range expansion of apomictic plants after climate changes, has been described for Northern Hemisphere gametophytic apomicts. But similar trends have been observed for sporophytic apomicts of Cerrado, the savannas in Brazil. Eriotheca pubescens is a common Cerrado tree, an agamic complex of either hexaploid/polyembryonic apomicts or tetraploid/monoembryonic sexual individuals. Some populations have been described as a new species, Eriotheca estevesiae, all included in the Eriotheca Stellate Trichome Species Complex (ESTSC). Since breeding systems and ploidy are clearly associated with polyembryony and stomatal size, we used these ancillary features to map the reproductive and ploidy level traits of E. pubescens and E. estevesiae. Leaves and seeds were collected from individuals of 19 populations. Seeds were evaluated for the presence of polyembryony and leaves for stomatal measurements. Eight populations were monoembryonic while another eight were polyembryonic and for other three, the embryonic pattern was not readily verified. E. pubescens polyembryonic and hexaploid populations formed a homogeneous group, but monoembryonic plants were more variable. E. estevesiae populations were monoembryonic with smaller stomata. In contrast, some E. pubescens monoembryonic populations further south presented larger stomata. Despite these outliers, possibly mixed populations, stomatal size and embryonic pattern differed from northern to southern populations. Embryonic pattern and stomatal size indicated that northernmost populations of Eriotheca STSC (E. estevesiae) are diploid and sexual. Southernmost populations, mostly polyembryonic and with large stomata, are hexaploid and apomictic. This is in agreement with geographic parthenogenesis and range expansion of apomictic lineages to southern habitats available after the last glacial maximum.

Seedling development of Melochia pyramidata in different substrates and pot sizes / Desenvolvimento de mudas de Melochia pyramidataem diferentes substratos e tamanho de vasos

Native plants such asMelochia pyramidatahave been targeted in various scientific studies due to their potential uses. From this perspective, the present study aimed to separately evaluate the effect of different substrates and pot sizeson the production of seedlings of M. pyramidatagenotypes propagated by cuttings. Theexperimentwas conducted in a plant nursery at the Center of Agricultural Sciences of theFederal University of Paraíba, Areia, PB, Brazil. Theexperimental design was completely randomizedandarranged in a 3 x 2factorialdesignwith three substrate types (sand, commercial substrate Plantmax®, andsand+ manure) and two genotypes(MP1 andMP2), withfive replications. Pot size evaluation used a 2 x 2factorial designwith two pot sizes(V1: 1.8 LandV2: 0.9 L), two genotypes(MP1 andMP2), andfour replications. The evaluations began seven days after the cuttings were planted in tubes and continued weekly for two months by evaluating the number of sprouts and leaves. After two months, these seedlings were transplanted to pots and evaluated for the number of sprouts, leaves, fruits, flower buds, leaf width, and leaf length. The data were subjected to analysis of variance and test of means(Tukey) at5% probability. The cuttings ofM. pyramidatadeveloped best in washed sand and in the commercial substratePlantmax®. The genotypes developed best in 1.8-L pots, showing more leaves, flower buds, and fruits.(AU)

As plantas nativas, como a Melochia pyramidata, têm sido alvos de vários estudos científicos devido suas potencialidades de usos. O objetivo deste trabalho foi avaliar, separadamente, o efeito de substratos e tamanhos de vasos na produção de mudas de genótipos de M. pyramidata pelo método da estaquia. O experimento foi conduzido em casa de vegetação no Centro de Ciências Agrárias da Universidade Federal da Paraíba, Areia, PB, Brasil. O delineamento experimental foi inteiramente casualizado, em esquema fatorial 3 x 2, com três tipos de substratos (areia, substrato comercial Plantmax® e areia + esterco) e 2 genótipos (MP1 e MP2.), com 5 repetições. Para a avaliação feita em diferentes tamanhos de vasos, utilizou-se o esquema fatorial 2 x 2, com 2 tamanhos de vasos (V1: 1,8 Litros e V2: 0,9 Litros) e 2 genótipos (MP1 eMP2), com 4 repetições. As avaliações iniciaram-se após sete dias do plantio das estacas nos tubetes, e continuaram, semanalmente, por dois meses, avaliando o número de brotações e o número de folhas. Após os dois meses, estas mudas foram transplantadas para vasos, onde avaliou número de brotações, número de folhas, número de frutos, número de botões florais, largura da folha e comprimento da folha. Os dados foram submetidos à análise de variância e teste de médias (Tukey) a 5% de probabilidade. As estacasde M. pyramidata apresentaram melhor desenvolvimento em areia lavada e substrato comercial Plantmax®. Os genótipos se desenvolveram melhor em vasos de 1,8 litros, com maiores quantidades de folhas, botões florais e frutos.(AU)

Nuclear phylogeography of the temperate tree species Chiranthodendron pentadactylon (Malvaceae): Quaternary relicts in Mesoamerican cloud forests.

BACKGROUND: The Mexican hand tree or Canac (Chiranthodendron pentadactylon) is a temperate tree species of cloud and pine-oak forests of southern Mexico and Guatemala. Its characteristic hand-shaped flower is used in folk medicine and has constituted the iconic symbol of the Sociedad Botánica de México since 1940. Here, the evolutionary history of this species was estimated through phylogeographic analyses of nuclear DNA sequences obtained through restriction site associated DNA sequencing and ecological niche modeling. Total genomic DNA was extracted from leaf samples obtained from a representative number (5 to 10 per sampling site) of individuals distributed along the species geographic range. In Mexico, population is comprised by spatially isolated individuals which may follow the trends of cloud forest fragmentation. By contrast, in Guatemala Chiranthodendron may constitute a canopy dominant species near the Acatenango volcano. The distributional range of this species encompasses geographic provinces separated by the Isthmus of Tehuantepec. The objectives of the study were to: (i) estimate its genetic structure to define whether the observed range disjunction exerted by the Isthmus of Tehuantepec translates into separate populations, (ii) link population divergence timing and demographic trends to historical climate change, and (iii) test hypotheses related to Pleistocene refugia. RESULTS: Patterns of genetic diversity indicated high levels of genetic differentiation between populations separated by the Isthmus. The western and eastern population diverged approximately 0.873 Million years ago (Ma). Demographic analyses supported a simultaneous split from an ancestral population and rapid expansion from a small stock approximately 0.2 Ma corresponding to a glacial period. The populations have remained stable since the LIG (130 Kilo years ago (Ka)). Species distribution modelling (SDM) predicted a decrease in potential distribution in the Last Interglacial (LIG) and an increase during the Last Glacial Maximum (LGM) (22 Ka), Mid-Holocene (6 Ka) and present times. CONCLUSIONS: Divergence time estimations support the hypothesis that populations represent Quaternary relict elements of a species with broader and northernmost distribution. Pleistocene climatic shifts exerted major influence on the distribution of populations allowing dispersion during episodes of suitable climatic conditions and structuring during the first interglacial with a time period length of 100 Kilo years (Kyr) and the vicariant influence of the Isthmus. Limited demographic expansion and population connectivity during the LGM supports the moist forest hypothesis model.

Simultaneous selection of cupuassu tree and Brazilian mahogany genotypes in an agroforestry system in Pará state, Brazil / Seleção simultânea de genótipos de cupuaçuzeiro e mogno brasileiro, em um sistema agroflorestal no Pará, Brasil

This study aimed to evaluate and select progenies and matrices of cupuassu-tree siblings, and to select Brazilian mahogany matrices with superior characteristics to be used in agroforestry systems (AFS). Twenty-five full cupuassu tree sibling progenies and one Brazilian mahogany half-sibling progeny were evaluated. The study was conducted for 14 years in a commercial property in Tomé Açu, Pará State, Brazil. The number of fruits and fruit yield per plant, as well as the rate of plants with symptoms of witches broom disease, were used as response variables for cupuassu. For mahogany, the following response variables were used: total height, commercial height, crown height, diameter at breast height and commercial wood volume. There were five cupuassu tree progenies with interesting characteristics to be used in AFS. Based on the high values observed in the selection accuracy and heritabilities, a good potential for the selection of promising individuals in the cupuassu tree population is inferred. Based on the ranking of genotypic values and their agronomic performance, 10 matrices of cupuassu tree were selected. For Brazilian mahogany, three matrices showed excellent silvicultural performance. The matrices of these two species should be propagated vegetatively in order to evaluate clonal tests.(AU)

Este trabalho teve por objetivo avaliar e selecionar progênies e matrizes de irmãos completos de cupuaçuzeiro e, simultaneamente, selecionar matrizes de mogno brasileiro com características superiores, para utilização em sistemas agroflorestais (SAFs). Foram avaliadas 25 progênies de irmãos completos de cupuaçuzeiro e uma progênie de meio irmão de mogno brasileiro. O estudo foi conduzido por 14 anos em uma propriedade comercial em Tomé Açu, Pará. Para o cupuaçuzeiro, foram utilizadas como variáveis de resposta o número e a produção de frutos por planta, e a taxa de plantas com sintomas da doença vassoura-de-bruxa. Para o mogno, empregou-se a altura total, altura comercial, altura da copa, DAP e volume de madeira comercial. Os resultados revelaram cinco progênies de cupuaçuzeiro com características interessantes para emprego em SAFs. Com base nos altos valores observados na acurácia de seleção e herdabilidade, inferimos um bom potencial de seleção de indivíduos promissores na população de cupuaçuzeiro. Dez matrizes de cupuaçuzeiro foram selecionadas com base no ranqueamento dos valores genotípicos e desempenho agronômico. Para o mogno brasileiro, três matrizes apresentaram ótimo desempenho silvicultural. As matrizes destas duas espécies deverão ser propagadas vegetativamente, para avaliação em ensaios clonais.(AU)

First Record of Mansonia dyari From Saint Croix, United States Virgin Islands.

The first report of Mansonia dyari on Saint Croix, United States Virgin Islands (USVI), is confirmed. Adult and larval specimens were collected in 2018 and 2019 through adult surveillance and larval collections. Specimens were identified by microscopic methods, and a representative specimen was confirmed by DNA sequencing (mitochondrial cytochrome c oxidase subunit I). Morphological features are reviewed and compared with Mansonia flaveola, a species previously reported in the USVI. Notes are provided on the locations, collection methods, and mosquito associates found with Ma. dyari in the USVI.

Genetic, evolutionary and phylogenetic aspects of the plastome of annatto (Bixa orellana L.), the Amazonian commercial species of natural dyes.

MAIN CONCLUSION: The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.

Phenotypic and genotypic characterization and compatibility among genotypes to select elite clones of cupuassu / Caracterização fenotípica, genotípica e compatibilidade entre genótipos para seleção de clones elite de cupuaçuzeiro

The expansion of the genetic base of cultivated materials is an ongoing activity of the cupuassu (Theobroma grandiflorum) breeding program. However, the parents involved need to be genotypically and phenotypically characterized to ensure compatibility of crossings, as well as to assist in the selection of more promising individuals for hybridization. This study aimed to identify and select T. grandiflorum clones that are compatible and genetically divergent using tools such as the estimates of genotypic, phenotypic, and combined distances, as well as the compatibility rates among clones. The genetic distance analysis of the clones was performed with 14 heterologous microsatellite primers of cocoa (Theobroma cacao) that amplify the DNA of cupuassu. Phenotypic characterization was based on 14 variables related to fruit production. The joint dissimilarity matrix was obtained by means of the sum of the phenotypic and molecular dissimilarity matrices. The intra- and inter-clonal compatibility was estimated through controlled crossings. A low correlation was noted between the dissimilarity matrices based on the molecular and agronomic data. As for compatibility, all clones were self-incompatible, with different compatibility rates when crossed. The compatibility index was strongly influenced by the degree of relationship of the clones. It was possible to identify and select the most promising sets of cupuassu clones to be used in breeding programs, despite their genetic relationship.(AU)

A ampliação da base genética dos materiais de cultivo é trabalho contínuo do programa de melhoramento genético do cupuaçuzeiro (Theobroma grandiflorum). Há necessidade, entretanto, que os parentais envolvidos estejam caracterizados fenotípica e genotipicamente, para auxiliar na escolha dos indivíduos que serão hibridizados, bem como para garantir os mecanismos de rastreabilidade e proteção das cultivares. Este estudo teve como objetivo identificar e selecionar clones de cupuaçuzeiro inter-compatíveis e geneticamente divergentes, utilizando como ferramentas as distâncias genotípica, fenotípica e combinada mistas, assim como as taxas de compatibilidade entre os clones. Estimativas das distâncias genéticas entre os clones foram realizadas com base em 14 iniciadores microssatélites heterólogos de cacaueiro (Theobroma cacao) que amplificam o DNA do cupuaçuzeiro. Para a caracterização fenotípica foram empregadas 14 variáveis relacionadas à produção de frutos. A matriz de dissimilaridade conjunta foi obtida por meio da soma das matrizes de dissimilaridade fenotípica e molecular. A compatibilidade intra e inter-clonal foi estimada através de cruzamentos controlados. Houve uma baixa correlação entre as matrizes de dissimilaridade com base nos dados moleculares e agronômicos. Quanto à compatibilidade, todos os clones foram auto-incompatíveis, contudo, compatíveis entre si, com diferentes taxas. O índice de compatibilidade foi fortemente influenciado pelo grau de relacionamento dos clones. Foi possível identificar e selecionar os conjuntos de clones de cupuaçuzeiro mais promissores para ser usados em melhoramento genético, apesar da ocorrência de relação genética entre eles.(AU)

Simulating the selfing and migration of Luehea divaricata populations in the Pampa biome to investigate the conservation potential of their genetic resources.

Computer simulations are an important tool for developing conservation strategies for forest species. This study used simulations to investigate the genetic, ecological, and reproductive patterns that contribute to the genetic structure of the tree Luehea divaricata Mart. & Zucc. in five forest fragments in the Brazilian Pampa biome. Using the EASYPOP model, we determined the selfing and migration rates that would match the corresponding genetic structure of microsatellite marker data (based on observed and expected heterozygosity parameters). The simulated reproductive mode was mixed, with a high rate of outcrossing (rate = 0.7). This was consistent with a selfing-incompatible system in this species, which reduced, but did not prevent, selfing. The simulated migration rate was 0.02, which implied that the forest fragments were isolated by distance, and that the inbreeding coefficients were high. Based on Nei's gene diversity analysis, 94% of the genetic variability was distributed within the forest fragments, and only 6% of the genetic diversity was caused by differences between them. Furthermore, the minimum viable population and minimum viable area genetic conservation parameters (which determine conservation potential in the short and long term) suggested that only the Inhatinhum forest fragment had the short-term potential to maintain its genetic diversity. However, in the long term, none of the forest fragments proved to be sustainable, indicating that the populations will require intervention to prevent a decline in genetic variability. The creation of ecological corridors could be a useful solution to connect forest fragments and enhance gene flow between them.

Diversity and spatial genetic structure of a natural population of Theobroma speciosum (Malvaceae) in the Brazilian Amazon / Diversidad y estructura genética espacial de una población natural de Theobroma speciosum (Malvaceae) en la Amazonía brasileña

Abstract:The quantification of genetic diversity and intrapopulation spatial genetic structure (SGS) of tree species are important aspects for in and ex situ conservation practices. In this study we seek to understand the importance of conservation areas by quantifying the genetic diversity and the spatial genetic structure of a natural population of Theobroma speciosum. Within this population, 49 adults and 51 subadults were genotyped for five microsatellite loci. The results showed that adults and subadults have similar levels of genetic diversity and inbreeding (adults: A= 10.4, Ae = 10.3, F= 0.68, subadults: A= 10.6, Ae= 10.6, F= 0.57). Genetic diversity was spatially structured within the population, and the results suggest that near-neighbor trees up to a distance of 70 m are likely related. SGS is likely the result of short-distance seed dispersal, the short-distance range of pollinators, and infrequent breaches of the self-incompatible mating system. Considering the high demographic density of the species and size of the study area, as well as the high average number of alleles per locus and the presence of rare alleles, we believe that the study population is an excellent resource for in situ genetic conservation of T. speciosum. The study area is also a useful resource for collecting germplasm for ex situ conservation and seed collection, either for breeding programs used in the restoration of degraded areas or forest improvement. Rev. Biol. Trop. 64 (3): 1091-1099. Epub 2016 September 01.

ResumenLa cuantificación de la estructura de la diversidad genética e genética intrapoblacional espacial (SGS) de especies de árboles son importantes en las prácticas de conservación ex situ. En este estudio se busca determinar la importancia de las unidades de conservación mediante la cuantificación de la diversidad genética y la estructura espacial de una población natural de Theobroma speciosum. Dentro de esta población, a 49 adultos y 51 subadultos se les analizó el genotipo para cinco microsatélites loci. Los resultados mostraron que los adultos y subadultos tienen niveles similares de diversidad genética y endogamia (adultos: A= 10.4, Ae = 10.3, F= 0.68; subadultos: A= 10.6, Ae = 10.6, F= 0.57). La diversidad genética detectada fue estructurada espacialmente dentro de la población y los árboles vecinos hasta una distancia de 70 m estaban probablemente relacionados. Esta SGS es probablemente el resultado de la dispersión de semillas de corta distancia. El establecimiento de unidades de conservación permanentes como el Parque Nacional Juruena mostró que las áreas de conservación son herramientas valiosas en la preservación de la diversidad genética en las poblaciones naturales. Debido a la alta densidad demográfica de la especie y el tamaño del área de estudio, así como el elevado número promedio de alelos por locus y la presencia de alelos raros, creemos que la población de estudio es un excelente recurso para la conservación genética in situ de T. speciosum y también es un recurso útil de germoplasma para la recolección de conservación ex situ y la semilla, ya sea para programas de mejoramiento utilizados en la restauración de áreas degradadas o mejora de los bosques.

Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum.

Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality-selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality.

Diversity and spatial genetic structure of a natural population of Theobroma speciosum (Malvaceae) in the Brazilian Amazon.

The quantification of genetic diversity and intrapopulation spatial genetic structure (SGS) of tree species are important aspects for in and ex situ conservation practices. In this study we seek to understand the importance of conservation areas by quantifying the genetic diversity and the spatial genetic structure of a natural population of Theobroma speciosum. Within this population, 49 adults and 51 subadults were genotyped for five microsatellite loci. The results showed that adults and subadults have similar levels of genetic diversity and inbreeding (adults: A= 10.4, Ae = 10.3, F= 0.68, subadults: A= 10.6, Ae= 10.6, F= 0.57). Genetic diversity was spatially structured within the population, and the results suggest that near-neighbor trees up to a distance of 70 m are likely related. SGS is likely the result of short-distance seed dispersal, the short-distance range of pollinators, and infrequent breaches of the self-incompatible mating system. Considering the high demographic density of the species and size of the study area, as well as the high average number of alleles per locus and the presence of rare alleles, we believe that the study population is an excellent resource for in situ genetic conservation of T. speciosum. The study area is also a useful resource for collecting germplasm for ex situ conservation and seed collection, either for breeding programs used in the restoration of degraded areas or forest improvement.

Genetic structure of natural populations of Theobroma in the Juruena National Park, Mato Grosso State, Brazil.

Several species within the genus Theobroma have particularly high economic value, including T. cacao and T. grandiflorum. Other species in this genus, such as T. speciosum and T. subincanum, have potential value for use in the conservation of genetic diversity in breeding programs. These latter species could also be domesticated or improved to produce commercial products. Using 13 simple sequence repeat loci, the population structure and genetic diversity of T. speciosum and T. subincanum natural populations in the Juruena National Park, Mato Grosso State, Brazil, was studied. We sampled all individuals of each species (N = 25) present inside a designated research area established by the Program for Research on Biodiversity. The average number of alleles per locus was 5 for T. speciosum and 6.69 for T. subincanum, with average PIC values above 0.5 in both species. All evaluated individuals varied genetically. Seeds from the individuals analyzed will be useful for the development of germplasm banks and for establishment of breeding programs.

Historical gene flow within and among populations of Luehea divaricata in the Brazilian Pampa.

Within and among population gene flow is a central aspect of the evolutionary history of ecosystems and essential for the potential for adaptive evolution of populations. We employed nuclear microsatellite markers to assess inter- and intra-population gene flow in five natural populations of Luehea divaricata growing in the Pampa biome, in southern Brazil. This species occurs in practically all secondary forests of the Pampa and has recognized ecological significance for these formations. The genetic structuring of the studied populations suggests limited gene dispersal among forest fragments, despite a homogeneous level of migration among populations. Notwithstanding the gene flow among populations, significant SGS is still found in some fragments. Significant spatial genetic structure within population was also found likely as result of limited seed and/or pollen dispersal. The scattered distribution of the populations and their relatively high density seem to limit pollen dispersal. Also seed dispersal by wind is not efficient due to large distances among forest formations. As conservationist actions towards preserving the genetic resources of L. divaricata and the Brazilian Pampa, we suggest the protection of the existing forest formations and the maintenance of the natural expansion of the forests over the grasslands in the biome.

Remnant Pachira quinata pasture trees have greater opportunities to self and suffer reduced reproductive success due to inbreeding depression.

Habitat fragmentation is extensive throughout the world, converting natural ecosystems into fragments of varying size, density and connectivity. The potential value of remnant trees in agricultural landscapes as seed sources and in connecting fragments has formed a fertile area of debate. This study contrasted the mating patterns of bat-pollinated Pachira quinata trees in a continuous forest to those in pasture through microsatellite-based paternity analysis of progeny. The breeding system was determined by analysis of pollen tube growth and seed production from controlled pollinations. Fitness of selfed and outcrossed seed was compared by germination and seedling growth. There was more inbreeding within pasture trees (outcrossing=0.828±0.015) compared with forest trees (0.926±0.005). Pasture trees had fewer sires contributing to mating events, but pollen dispersal distances were greater than those in the forest. Paternity analysis showed variation in outcrossing rates among pasture trees with high proportions of external and self pollen sources detected. A leaky self-incompatibility system was found, with self pollen having reduced germination on stigmas and slower growth rate through the style. Controlled pollinations also showed a varied ability to self among trees, which was reflected in the selfing rates among pasture trees shown by the paternity analysis (0-80% selfing). Self pollination resulted in lower seed set, germination and seedling growth compared with outcrossing. While remnant trees in agricultural landscapes are involved in broader mating patterns, they show increased but varied levels of inbreeding, which result in reduced fitness.

Do chromosome numbers reflect phylogeny? New counts for Bombacoideae and a review of Malvaceae s.l.

UNLABELLED: • PREMISE OF THE STUDY: Whole genome duplication (WGD) and specific polyploidy events marked turning points for angiosperm genome structure and evolution. Therefore, cytogenetic studies of polyploidy-prone groups such as the tropical Malvaceae and plant formations such as as the Brazilian Cerrado have gained further importance. We present new chromosome counts for Cerrado Bombacoideae and revised chromosome numbers for the Malvaceae s.l., compare these between subfamilies, and relate them to phylogenetic signal.• METHODS: We studied the chromosome number of Eriotheca candolleana, E. gracilipes, E. pubescens, Pachira glabra, Pseudobombax longiflorum, and P. tomentosum. We also compared Eriotheca species ploidy levels using flow cytometry. We compiled chromosome numbers for 557 species of Malvaceae s.l., including 37 Bombacoideae species. We included this information in a phylogenetic reconstruction based on chloroplast matK-trnK DNA to evaluate chromosome evolution of the Malvaceae s.l. and the Bombacoideae in particular.• KEY RESULTS: The Cerrado Bombacoideae presented consistently high chromosome numbers. Numbers for Eriotheca species were among the highest and varied among populations. Flow cytometry analyses showed similar 1Cx DNA for all cytotypes and indicated neopolyploidy. Chromosome numbers differed between subfamilies, with the lowest numbers in the Malvoideae and Byttnerioideae and the highest in Tilioideae. Chromosome numbers had significant phylogenetic signal for Bombacoideae but not for Malvoideae or Malvaceae s.l.• CONCLUSIONS: Clearly distinct chromosome numbers allied to monophyly provide some support for a circumscription of the Bombacoideae and distinction within the Malvaceae. The phylogenetic signal for chromosome number supports the idea of an ancient WGD and further neopolyploidy events as important evolutionary trends for the Bombacoideae.

Pollen and stomata morphometrics and polyploidy in Eriotheca (Malvaceae-Bombacoideae).

Approximately 70% of the angiosperm species are polyploid, an important phenomenon in the evolution of those plants. But ploidy estimates have often been hindered because of the small size and large number of chromosomes in many tropical groups. Since polyploidy affects cell size, morphometric analyses of pollen grains and stomata have been used to infer ploidy level. Polyploidy is present in many species of the Cerrado, the Neotropical savanna region in Central Brazil, and has been linked to apomixis in some taxa. Eriotheca gracilipes and Eriotheca pubescens are common tree species in this region, and present cytotypes that form reproductive mosaics. Hexaploid individuals (2n = 6x = 276) are polyembryonic and apomictic, while tetraploid and diploid individuals (2n = 2x = 92, 2n = 4x = 184) are sexual and monoembryonic. We tested whether morphometric analysis can be used to estimate ploidy levels in E. gracilipes and E. pubescens individuals. Pollen material from diploid and hexaploid individuals of E. gracilipes, and tetraploid and hexaploid individuals of E. pubescens, were fixed in 50% FAA, and expanded leaves were dried in silica gel. Pollen grains and stomata of at least five individuals from each population were measured. The results demonstrate that all measures were significantly different among cytotypes. Individuals with higher levels of ploidy (hexaploid) all presented measurements that were higher than those with lower levels (diploid and tetraploid). There was no overlap between ploidy levels in each species at 95% confidence interval. Thus, the size of the pollen grains and stomata are effective parameters for analysis of ploidy levels in E. gracilipes and E. pubescens.

Genetic diversity in natural populations of Theobroma subincanum Mart. in the Brazilian Amazon.

The genus Theobroma, recently reclassified in the family Malvaceae, comprises some species with high economic potential, including the cupuí, Theobroma subincanum Mart., which has not yet been domesticated, and whose genetics and population structure are mostly unknown. This study aimed to assess the population structure and genetic diversity in natural populations of T. subincanum Mart., using inter-simple sequence repeat (ISSR) markers. A total of 59 individuals were sampled in three geographically separate populations, CFA, CMN, and CPT. Nei's genetic distance was estimated to characterize populations with the use of 13 polymorphic primers. The analysis of molecular variance revealed that the variability between populations (51.71%) was higher than that within populations (48.29%). Among the three populations, CPT showed the highest diversity index and percentage of polymorphism. The ISSR molecular markers were efficient and presented sufficient polymorphism to estimate genetic diversity in populations of T. subincanum Mart.

Genetic structure of the Atlantic Rainforest tree species Luehea divaricata (Malvaceae).

The Atlantic Rain Forest is one of the most important Brazilian biomes and a hotspot for biodiversity that is characterized by its high level of endemism, where new species are still being described. Luehea divaricata (Malvaceae) is commonly found in riparian forests areas of the Atlantic forest. Because of the importance of this species in reforestation programs, we used nine pairs of microsatellite loci to study the genetic variability of this species along its distribution area and verify if fragmentation is compromising the survival of these populations. A total of 50 alleles were obtained with an average observed and expected heterozygosity of 0.53 and 0.67, respectively. Seven of the nine populations studied showed a heterozygosity deficit. Most of the genetic diversity was found within populations; while the level of genetic differentiation was moderated (6.84) between populations. Different levels of gene flow between the populations were detected. Positive and significant values of Fis were found for seven populations. The signal test for excess of heterozygosity indicated that a recent genetic bottleneck occurred in the fragmented populations. The dendrogram constructed by the UPGMA method revealed the formation of seven clusters, which was confirmed by the Bayesian analysis for number of K clusters. The presence of several pairs of loci in linkage disequilibrium confirms that these populations experienced a loss of genetic diversity caused by genetic drift. The results showed that it is necessary to develop management strategies for the conservation of these populations of L. divaricata as the viability of the next generations are severely compromised.