Spondias mombin (Anarcadiaceae): molecular characterization and conservation / Spondias mombin (Anarcadiaceae): caracterización molecular y conservación

Abstract Introduction: The fruit of the yellow mombin (Spondias mombin L.) is notable due to its sensory and functional qualities. However, there is little knowledge regarding the genetic diversity of this species, and this would aid the implantation of the cultivation of the fruit as a crop, since current production is based on extractivism. Objective: Evaluate the diversity and genetic structure of natural populations of S. mombin in the state of Mato Grosso, Brazil, through microsatellite molecular markers in order to assist in the implementation of conservation strategies and the collection of genetic resources. Methods: A total of 139 S. mombin individuals were sampled in ten natural populations. PCR amplifications were performed with seven fluorescence-marked microsatellite primers. Genetic diversity was evaluated by the number of alleles, expected (He) and observed heterozygosity (Ho), polymorphic information content (PIC), fixation index (ƒ), rare and exclusive alleles. The genetic structure was evaluated using AMOVA, UPGMA dendrogram and Bayesian statistical analysis. Results: 46 alleles were amplified, which had an average of 6.6 alleles per locus. He was higher than Ho and f was positive, indicating the presence of inbreeding. The PIC ranged from 0.048 to 0.700, and only two loci were poorly informative. We found 27 rare alleles and 16 unique alleles. The largest component of variation was intrapopulational (90.64 %). The estimated gene flow was 1.99, which indicates that there is no genetic isolation between populations, and justifies the FST value (0.0963). The ten populations were grouped into two groups, and two populations constituted an isolated group. The Mantel test demonstrated that the genetic structure is not related to the geographic distance between populations. Conclusion: There is genetic diversity in the populations of S. mombin, which must be conserved in situ or ex situ, due to the diversity they present and because they are promising sources for collection of germplasm.

Resumen Introducción: El fruto amarillo del jobo o yuplón (Spondias mombin L.) destaca por sus cualidades sensoriales y funcionales. Sin embargo, existe poco conocimiento sobre la diversidad genética de esta especie, lo que ayudaría a la implantación del cultivo del fruto como cultivo, ya que la producción actual se basa en el extractivismo. Objetivo: Evaluar la diversidad y estructura genética de poblaciones naturales de S. mombin en el estado de Mato Grosso, Brasil, a través de marcadores moleculares microsatélites para ayudar en la implementación de estrategias de conservación y recolección de recursos genéticos. Métodos: Se muestrearon un total de 139 individuos de S. mombin en diez poblaciones naturales. Las amplificaciones por PCR se realizaron con siete cebadores de microsatélites marcados con fluorescencia. La diversidad genética se evaluó por el número de alelos, heterocigosidad esperada (He) y observada (Ho), contenido de información polimórfica (PIC), índice de fijación (ƒ), alelos raros y exclusivos. La estructura genética se evaluó mediante AMOVA, dendrograma UPGMA y análisis estadístico bayesiano. Resultados: Se amplificaron 46 alelos, los cuales tenían un promedio de 6.6 alelos por locus. Fue más alto que Ho y f positivo, lo que indica la presencia de endogamia. El PIC osciló entre 0.048 y 0.700, y solo dos loci fueron poco informativos. Encontramos 27 alelos raros y 16 alelos únicos. El mayor componente de variación fue intrapoblacional (90.64 %). El flujo de genes estimado fue de 1.99, lo que indica que no hay aislamiento genético entre poblaciones y justifica el valor de FST (0.0963). Las diez poblaciones se agruparon en dos grupos y dos poblaciones constituyeron un grupo aislado. La prueba de Mantel demostró que la estructura genética no está relacionada con la distancia geográfica entre poblaciones. Conclusión: Existe diversidad genética en las poblaciones de S. mombin, la cual debe ser conservada in situ o ex situ, por la diversidad que presentan y porque son fuentes promisorias para la recolección de germoplasma.

Diversidade genética estimada com marcadores entre sequências simples repetidas em cultivos comerciais de Cupuaçuzeiro / Genetic diversity estimated using inter-simple sequence repeat markers in commercial crops of cupuassu tree

RESUMO:Quinze primers ISSR (entre sequências simples repetidas) foram utilizados para avaliar a diversidade genética entre e dentro de pomares comerciais de Theobroma grandiflorum (Willd. ex Spreng.) K. Schum. Para isso, foram analisados sessenta indivíduos, distribuídos nos três cultivos. Um total de 102 bandas foi amplificado, com uma porcentagem de 52,0% de polimorfismo em nível de espécie e média de 6,8 alelos por primer ISSR. A média do Índice de Conteúdo Polimórfico (PIC) foi de 0,55. Em relação aos índices de diversidade gênica de Nei (H) e de Shannon (I), os cultivos analisados apresentaram os valores: SAR H = 0,114 e I = 0,177; SSL H = 0,108 e I = 0,162 e SEC H = 0,104 e I = 0,156, considerados valores de moderados a baixos. A AMOVA revelou 34,91% da variância total entre os cultivos e 65,09% dentro deles. Os marcadores moleculares ISSR revelaram que há diversidade genética dentro de cada cultivo comercial estudado, portanto é possível selecionar genótipos superiores que poderão ser utilizados para originar cultivos mais uniformes. Esse resultado tem sido considerado de grande relevância, por fornecer ferramentas para a implementação de programas de melhoramento e delineamento de estratégias de conservação ex situ e in situ.

ABSTRACT:Fifteen ISSR (inter-simple sequence repeat) primers were used to evaluate the genetic diversity among and within commercial crops of T. grandiflorum (Willd. ex Spreng.) K. Schum. For this, 60 specimens were analyzed, distributed in three crops. A total of 102 bands were amplified, with a polymorphism percentage of 52.0% at species level and an average of 6.8 alleles per ISSR primer. The average for polymorphism information content (PIC) index was 0.55. In relation to the genetic diversity index of Nei (H), and Shannon (I), crops analyzed showed the following values: : SAR H = 0,114 e I = 0,177; SSL H = 0,108 e I = 0,162 e SEC H = 0,104 e I = 0,156, considered moderate to low values. AMOVA showed 34.91% of total variance among the crops, and 65.09% within them. The ISSR molecular markers revealed that there is genetic diversity within each commercial crops studied, thus is possible to select superior genotypes that can be used to give more uniform crops. This result has been considered of great relevance, to provide tools for breding implementation programs and design conservation strategies ex situ and in situ.

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.

Use of anatomical root markers for species identification in Catasetum (Orchidaceae) at the Portal da Amazônia region, MT, Brazil / Uso de marcadores anatômicos radiculares para a determinação de espécies de Catasetum (Orchidaceae) da Região do Portal da Amazônia, MT, Brasil

Orchidaceae is one of the largest botanical families, with approximately 780 genera. Among the genera of this family, Catasetum currently comprises 166 species. The aim of this study was to characterize the root anatomy of eight Catasetum species, verifying adaptations related to epiphytic habit and looking for features that could contribute to the vegetative identification of such species. The species studied were collected at the Portal da Amazônia region, Mato Grosso state, Brazil. The roots were fixed in FAA 50, cut freehand, and stained with astra blue/fuchsin. Illustrations were obtained with a digital camera mounted on a photomicroscope. The roots of examined species shared most of the anatomical characteristics observed in other species of the Catasetum genus, and many of them have adaptations to the epiphytic habit, such as presence of secondary thickening in the velamen cell walls, exodermis, cortex, and medulla. Some specific features were recognized as having taxonomic application, such as composition of the thickening of velamen cell walls, ornamentation of absorbent root-hair walls, presence of tilosomes, composition and thickening of the cortical cell walls, presence of mycorrhizae, endodermal cell wall thickening, the number of protoxylem poles, and composition and thickening of the central area of the vascular cylinder. These traits are important anatomical markers to separate the species within the genus and to generate a dichotomous identification key for Catasetum. Thus, providing a useful tool for taxonomists of this group.

Orchidaceae é uma das maiores famílias botânicas, com cerca de 780 gêneros. Dentre seus gêneros, Catasetum inclui atualmente 166 espécies. Caracterizou-se a raiz de oito espécies de Catasetum com o objetivo de verificar caracteres relacionados ao hábito epifítico e contribuir para a taxonomia do grupo. As espécies foram coletadas na região do Portal da Amazônia, no norte do estado de Mato Grosso. Raízes foram fixadas em FAA 50 (1:1:8 formaldeído, ácido acético glacial e álcool etílico 50%), cortadas à mão livre e corados com azul de astra e fucsina. As ilustrações foram obtidas por meio do capturador de imagens acoplado ao fotomicroscópio. As raízes das espécies estudadas compartilharam a maioria dos caracteres anatômicos observados em outras espécies de Catasetum, e vários destes demonstraram adaptações ao hábito epifítico, tais como presença de espessamento secundário na parede das células do velame, da exoderme, do córtex e da medula. Alguns caracteres foram reconhecidos como tendo aplicação taxonômica, como composição do espessamento da parede das células do velame, ornamentação da parede dos pelos absorventes, presença de tilossomos, composição e espessamento da parede das células do córtex, presença de micorriza, tipo de espessamento da parede das células da endoderme, número de pólos de protoxilema e composição e tipo de espessamento da região central do cilindro vascular. Esses caracteres são importantes marcadores anatômicos, pois possibilitam separar as espécies dentro do gênero e gerar uma chave dicotômica de identificação para as Catasetum da região investigada, fornecendo, assim, uma ferramenta útil para os taxonomistas do grupo.

Molecular evidence of cryptic speciation, historical range expansion, and recent intraspecific hybridization in the Neotropical seasonal forest tree Cedrela fissilis (Meliaceae).

Molecular phylogeography can lead to a better understanding of the interaction between past climate events, large-scale vegetation shifts, and the evolutionary history of Neotropical seasonal forests. The endangered timber tree species Cedrela fissilis is associated with seasonal forests and occurs throughout South America. We sampled C. fissilis from 56 sites across the species' range in Brazil and Bolivia and obtained sequence data for nuclear and chloroplast DNA. Most specimens (149 out of 169) exhibited intraindividual polymorphism for the nuclear internal transcribed spacer (ITS). Cloning and an array of complementary sequence analyses indicated that the multiple copies of ITS were functional paralogs--concerted evolution in C. fissilis appeared to be incomplete. Independent Bayesian analyses using either ITS or cpDNA data revealed two separate phylogenetic lineages within C. fissilis that corresponded to populations located in separate geographic regions. The divergence occurred in the Early Pliocene and Late Miocene. We argue that climate-mediated events triggered dispersal events and split ancestral populations into at least two large refugial areas of seasonal forest that were located to the east and west of the present day Cerrado. Upon recent climate amelioration, formerly isolated lineages reconnected and intraspecific hybridization gave rise to intraindividual polymorphism and incomplete concerted evolution in C. fissilis.

Molecular phylogeography of Carapichea ipecacuanha, an amphitropical shrub that occurs in the understory of both semideciduous and evergreen forests.

The medicinal shrub Carapichea ipecacuanha (ipecac) is an amphitropic species with three disjunct areas of distribution. In the Brazilian Atlantic and Amazonian ranges, the species was associated mostly with the understory of seasonal semideciduous forests, whereas in the Central American-Colombian range, the species occurred in the understory of moist evergreen forests. We examined the phylogeographic structure of ipecac using chloroplast trnT-trnL and nuclear internal transcribed spacer (ITS) sequences from 120 and 46 specimens, respectively. To complement existing data on root alkaloid profiles, we used high-performance liquid chromatography to assess the levels of emetine and cephaeline in 33 specimens from the two Brazilian ranges. The three ranges shared neither nuclear nor chloroplast haplotypes. The phylogeographic structures showed an uneven distribution of genetic diversity, sharp breaks and high levels of genetic differentiation among ranges. Our results suggest that the extant populations are descendents of at least four distinct ancestral lineages. The Atlantic ipecacs showed higher levels of genetic diversity than ipecacs from the other two ranges; it is likely that they derive from two ancestral lineages, with long-term persistence in that region. The Amazonian ipecacs were monomorphic with respect to the ITS and cpDNA sequences, which supports the view that there was a recent expansion from a single parental source after a strong genetic bottleneck. The existence of a fourth distinct lineage is apparent from the high levels of genetic and chemical differentiation that we identified in the Central American-Columbian ipecacs.

Clonal diversity and conservation genetics of the medicinal plant Carapichea ipecacuanha (Rubiaceae).

The roots of the understorey shrub Carapichea ipecacuanha (ipecac) have medicinal properties, and the uprooting of wild plants has supplied most of the world demand for this species. Although under severe population decline, C. ipecacuanha lacks legal protection. In the wild, the aerial stems of ipecac clump together to form clusters with well-defined borders. Cluster size may range from several to hundreds of aerial stems. To investigate the extent of clonality among aerial stems in ipecac clusters, we sampled 50 wild clusters (a total of 291 aerial stems) and screened them with 89 inter-simple sequence repeat (ISSR) markers. The 291 aerial stems were grouped into 42 putative clones. The clonal groups generally consisted of aerial stems from the same cluster, and there was little or no genetic differentiation among aerial stems at the cluster level. These findings suggest that strategies designed to conserve ipecac in situ should not rely upon census data, which are based on the number of aerial stems per cluster and the number of clusters per population, because such data greatly underestimate the species effective population size and genetic diversity. Our results also indicate that this species needs protection at a federal level.

Clonal diversity and conservation genetics of the medicinal plant Carapichea ipecacuanha (Rubiaceae)

The roots of the understorey shrub Carapichea ipecacuanha (ipecac) have medicinal properties, and the uprooting of wild plants has supplied most of the world demand for this species. Although under severe population decline, C. ipecacuanha lacks legal protection. In the wild, the aerial stems of ipecac clump together to form clusters with well-defined borders. Cluster size may range from several to hundreds of aerial stems. To investigate the extent of clonality among aerial stems in ipecac clusters, we sampled 50 wild clusters (a total of 291 aerial stems) and screened them with 89 inter-simple sequence repeat (ISSR) markers. The 291 aerial stems were grouped into 42 putative clones. The clonal groups generally consisted of aerial stems from the same cluster, and there was little or no genetic differentiation among aerial stems at the cluster level. These findings suggest that strategies designed to conserve ipecac in situ should not rely upon census data, which are based on the number of aerial stems per cluster and the number of clusters per population, because such data greatly underestimate the species effective population size and genetic diversity. Our results also indicate that this species needs protection at a federal level.

Genetic diversity and geographic differentiation of disjunct Atlantic and Amazonian populations of Psychotria ipecacuanha (Rubiaceae).

Ipecac (Psychotria ipecacuanha) is a perennial, medicinal herb that grows in the understory of semi-deciduous tropical forests in the Neotropics. Ipecacs present a widely disjunct distribution, with two of its three ranges occurring in Brazil. The Amazonian populations are at least 1600 km from the nearest Atlantic populations. This work used ISSR markers to compare the genetic diversity and structure of populations from the two Brazilian ranges. Lower genetic diversity in Amazon populations (P = 60.11%, Hs = 0.18) and higher genetic diversity in Atlantic populations (P = 73.94%, Hs = 0.20) were detected. Differentiation between ranges were high (theta (B) = 0.6838, G(ST)-B = 0.6665). AMOVA revealed that 65.3% of the total molecular variance can be attributed to regional differences between the two ranges. Principal coordinate analyses and cluster analyses organized ipecacs at either individual or population level into two exclusive groups that correspond each to one of the two disjunct ranges, without exception. The results do not support a scenario that postulates human-mediated, long-distance dispersal events as a plausible origin for the distribution of the Brazilian ipecacs, but indicate geographic isolation as a long-standing barrier to genetic exchange and connectivity among populations from different ranges. Conservation implications are discussed.