Anadenanthera colubrina var. cebil is an important tree species for its cultural, economic, and medicinal uses in South America. In order to characterize A. colubrina populations, we collected fruits from four different sites (San ]]>
Anadenanthera colubrina var. cebil es una especie arbórea de importancia cultural, económica y medicinal en Sur América. Para estudiar las poblaciones de A. colubrina, recolectamos frutos de cuatro sitios diferentes ]]>
Palabras clave: biodiversidad, banco de germoplasma, marcadores moleculares, secuencia nucleotídica, conservación de especies vegetales. ]]>
Anadenanthera colubrina var. cebil (Vell.) Brenan belongs to the Fabaceae/Leguminosae family (Lewis, Schrire, MacKinder, & Lock, 2005), Mimosoideae subfamily. This tree species has economic, medicinal, and cultural applications in South America. It is widely distributed in Brazil, Paraguay, Bolivia and Argentina, where it is found in eleven provinces in the Northeast, Northwest and center, in mountain and transition forests and in Chaco ]]>
The characterization of germplasm collections allows the assessment of the variability of the preserved material, although this information is poorly documented in seed banks. When the characterizations are morphological, descriptors that exhibit high heritability are used due to their stability in different ]]>
Studies carried out on wild plant species have revealed that the distribution of morphological and chemical characteristics present patterns can be related to geographic regions (Nevo, Beiles, & Krugman, 1988; Nevo, Noy-Meir, Beiles, Krugman, & Agami, 1991). These variations may reflect the phenotypic plasticity of the individuals, genetic adaptations of the ]]>
Plantago was greater for morphological than for isoenzymatic markers, due to its great phenotypic plasticity in response to environmental differences. Makkar & Becker (1998) found that plants in semiarid environments contain more phenolic compounds than in humid African environments. Morphological differences were also reported in relation to genetic and environmental characteristics, such as soil moisture, ]]>
A. colubrina bark tannin content and environmental humidity.
Morphological characteristics are related to genetic traits, but this information is poorly documented. ]]>
A. colubrina was studied using micro-satellite markers, and polymorphisms were found in the studied populations (Feres et al., 2012), as well as in A. colubrina var. cebil inArgentina (Barrandeguy, Prinz, García, & Fin-keldey, 2012).
Different molecular markers have ]]>
Sequence comparison of the ITS region is widely used in taxonomy and molecular phylogeny. This can be explained by the relatively low evolutionary pressure acting on such non-functional sequences. For example, ITS has been proven especially ]]>
The aim of this work was to study the morphological descriptors and the genetic diversity of A. colubrina var. cebil, collected from different locations over its ]]>
Materials and Methods
Collection of fruits: Initially four populations of A. colubrina var. cebil (Ac) wereselected from different sites over the distribution area of the species in Salta province: El Gallinato (G), San Bernardo (B), El Cebilar (C) and Metán (M) (Fig. 1), all with different elevation and rainfall (Table 1). From each population, we collected mature fruits from 10 ]]>
Morphological approach of fruits and seeds: Morphological characterization was performed by randomly sampling 75 fruits from each population. We recorded the weight (Denver ]]>
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Isolation and sequencing of ITS-rDNA regions: Genomic DNA was extracted from the seeds of one tree from each population following the protocol of Doyle & Doyle (1990). PCR was performed according to Esteve-Zarzoso, Belloch, Uruburu, and Querol (1999). Briefly, the primers ITS1 (5´-TCC-GTAGGTGAACCTTGCGG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC -3´) were used for the PCR. The PCR mix contained 0.5μmol/L of each primer, 10μmol/L deoxy-nucleotides, ]]>
2 and 1×PCR buffer, 1 unit of Taq enzyme (AmpliTaq Gold® PCR Master Mix). The suspension was heated at 95°C for 5min in a PE 2400 thermocycler (Applied Biosystems, USA). PCR conditions were: 35 cycles of denaturing (94°C, 1min), annealing (55°C, 1min) and extension (72°C, 2min), followed by a final extension at 72°C for 10min. The fragments, ranging from 390-490nt. obtained by PCR, were purified by resin columns (Qiagen) and sent to sequencing to MWG company (Germany). ]]>
Nucleotide alignment and phylogenetic analysis: Multiple alignment of the ITS-rRNA region nucleotidic sequences was performed with Clustal W2 and Boxshade programs, manually refined (Thompson, Higgins, & Gib-son, 1994). A phylogenetic tree was generated by using the Gene Bee service, Tree-Top program choosing a Phylip format, with boot-strap values, in agreement with alignment (Saitou & Nei, 1987; Brodsky et al., 1995). Sequence accession numbers are AcBr (Brazilian): DQ787408; AcM: JQ910930; AcB: ]]>
Results
Morphological approach of fruits and seeds: The weight distributions of fruits were normal in the ]]>
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In the four populations, the proportion of plain seeds was low and ranged from 39% (Metán) to 55% (El Cebilar). The proportion of aborted seeds was high in all populations, although Metán had the highest level. The pro-portion of predated seeds was low and similar among populations (Table 2).
The analysis of the ]]>
Fig. 2). The distributions of seed weights were normal in El Cebilar and El Gallinato, whereas in San Bernardo and Metán, seed weight was skewed and negative leptokurtic. Seed weight varied between populations and was significantly higher in San Bernardo, lower in El ]]>
Table 3). The analysis of seed morphological descriptors showed that the more distant populations were San Bernardo and El Cebilar (Euclidean distance 4.09), and the closest were El Gallinato and El Cebilar. In addition, these two populations were more distant from San Bernardo than from Metán (Fig. ]]>
).
Genetic approach of A. colubrina var. cebil seeds: We isolated, for the first time, the ITS of rDNA from fresh seeds of four individual A. ]]>
var. cebil trees (one from each population) from Salta Province. They were long: 1 067, 960, 861, 580 base pairs, and were registered in GenBank. The sizes of the specificparts in the isolated regions of the individual trees were: ITS1=158bp; 5.8SrRNA=166bp; ITS2=170bp. We selected a restricted region of ITS, equal to San Bernardo (AcB) 450bp, El Cebilar (AcC) 448bp, El Gallinato (AcG) 448bp, Metán (AcM) 450bp, and aligned the nucleotidic sequences with a 397bp long sequence (AcBr, Brazilian), the only one found in Genbank, which was isolated from
A. colubrina in Brazil. The restricted regions contain the following parts: from nucleotide 1 to 95, the partial “ITS1”; from 96 to 261, the complete gene “5.8S rRNA”; from 262 to 450, the partial “ITS2”. The nucleotidic sequence comparison showed a high identity between San Bernardo (AcB) and Metán (AcM) with a similarity of 98.1%, as well as between the El Cebilar (AcC) and the El Gallinato (AcG) sequences. The identity decreased to 90.4% when the AcB sequence was compared with AcC and AcG. The Brazilian sequence showed a lower identi-ty, ranging from 83.6%, if compared with AcC and AcG, to 84.1% with AcB and 84.6% with AcM. ]]>
Then, we investigated the relationships among the ITS-rDNA regions of different individuals from ]]>
A. colubrina var. cebil. The nucleotidic alignment showed a very high conservation of the nucleotidic sequence among the different individuals (Fig. 4A).
Nevertheless, some differences were evident: transitions (among purine or among pyrimidines) and ]]>
Fig. 4A). These differences can be attributed to intraspecific polymorphism, perhaps due to the different environments in which the trees grew. ]]>
Figure 4B shows a graphic phylogram, with cluster algorithm and bootstrap values. In this phylogenetic tree, it is clear that Argentin-ian and Brazilian ITS genes form different branches; then, the Argentinian branch divides into two: one for AcB and AcM, and another giving rise to AcC and AcG. The Brazilian A. colubrina is an outgroup. The high bootstrap values ]]>
A. colubrina var. cebil has a similar percentage (ranging from 59 to 60%), while the Brazilian one presents a higher GC content (70%).
Discussion
The studies of phenotypic and genetic diversity, as well as the detection of local varieties adjusted to different environments, are crucial issues in long-term (in situ and ex situ) biodiversity conservation. They are also impor-tant for the ]]>
Although theoretical models suggest that balancing selection should be heavy on features related to biological fitness (McGinley, Temme, & Geber, 1987), numerous plant species have documented wide variations in the morphology of fruits and seeds also related to geographic variability (Baker, 1972; Busso & ]]>
In this work, we found variations in the morphological traits of fruits and seeds in the four studied populations of Argentinian A. colubrina var. cebil. The variation was recorded within populations, and it was higher in fruits than in seeds. ]]>
Regarding the genetic characterization, ITS sequences are used for phylogenetic studies due ]]>
It is possible to use gene trees to obtain information about species history. It is known that ITS-rDNA genes are good species evolution markers because they contain both conserved and variable regions. They are present and have similar functions in all organisms and are easy to clone (Ribeiro, Rapini, ]]>
A. colubrina var. cebil because of the few individual trees studied.
ITS region is a high variable region, so it is a particularly valuable resource for plant ]]>
In the present study, we used ITS-rDNA sequences to address one main objective: to reconstruct the phylogenetic relationships among the four individuals of A. colubrina var.cebil from Salta province. We isolated the ITS-rDNA from
A. colubrina var. cebil, and we found variability within the species. The highest similarity of nucleotidic sequence was found between San Bernardo and Metán, and between El Cebilar and El Gallinato, as demonstrated by phylogenetic analysis. This is in agreement with our analysis of seed morphological descriptors. The only sequence recorded for the same species, from a plant in Brazil, presented a lower similarity to all Argentinian sequences, suggesting a divergent evolution. Furthermore, GC content was found to be different from the ]]>
Even though the present genetic study is limited solely to A. colubrina var. cebil species within a ]]>
Sarracenia purpurea. Our results agree with the concept that there is great plant phenotypic plasticity that allows the assessment of diversity in response to the environmental variations (Mondini, Noorani, & Pagnotta, 2009). Further studies are needed to explore this topic, through the investigation of a large number of ]]>
A. colubrina var. cebil species.
In this study, we concluded that the Argentinian tree A. colubrina var. cebil shows significant morphological and genetic variability and that ITS-rDNA molecular ]]>
The documentation and recording of phenotypic and genetic variability is crucial for the maintaining of the long-term conservation of biodiversity and for germplasm conservation.
In order to preserve ]]>
ex situ conservation purposes, it is important to collect seeds from many populations, especially for widespread species that grow in a variety of environments, such as A. colubrina var. cebil. However, further studies are needed to assess levels of variability within and between populations. A. colubrina ]]>
cebil is an important plant species because of its ornamental, medicinal, cultural, ecological and economic uses (wood, honey, alkaloids, tannin and gum extraction, restoration). These reasons point to the need for A. colubrina var. cebil conservation and propagation.
Acknowledgments
We thank the Research Council of the National University of Salta and INEAH for supporting this work and Maria Emilia Muñoz, Griselda Salas Barboza and Valeria Pastrana Ignes for the collaboration with the morphological characterization. We acknowledge Franco Palla for critical reading of the manuscript and for very helpful suggestions. ]]>
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*Correspondencia: Marta L. de Viana: Banco de Germoplasma de Especies Nativas, Instituto de Ecología y Ambiente Humano. Universidad Nacional de Salta. Avda. Bolivia 5150, 4400, Salta, Argentina; mldeviana@yahoo.com.ar Eugenia Giamminola: Banco de Germoplasma de Especies Nativas, Instituto de Ecología y Ambiente Humano. Universidad Nacional de Salta. Avda. Bolivia 5150, 4400, Salta, Argentina; eugeniagiamminola@yahoo.com.ar Roberta Russo: Istituto di Biomedicina e Immunologia Molecolare “A. Monroy”. Consiglio Nazionale delle Ricerche (CNR), Via Ugo La Malfa 153, 90146 Palermo, Italy; rrusso@ibim.cnr.it Mirella Ciaccio: Istituto di Biomedicina e Immunologia Molecolare “A. Monroy”. Consiglio Nazionale delle Ricerche (CNR), Via Ugo La Malfa 153, 90146 Palermo, Italy; ciaccio@ibim.cnr.it 1. Banco de Germoplasma de Especies Nativas, Instituto de Ecología y Ambiente Humano. Universidad Nacional de Salta. Avda. Bolivia 5150, 4400, Salta, Argentina; mldeviana@yahoo.com.ar, eugeniagiamminola@yahoo.com.ar 2. Istituto di Biomedicina e Immunologia Molecolare “A. Monroy”. Consiglio Nazionale delle Ricerche (CNR), Via Ugo La Malfa 153, 90146 Palermo, Italy; ciaccio@ibim.cnr.it, rrusso@ibim.cnr.it
Received 13-vi-2013. Corrected 20-X-2013. Accepted 22-Xi-2013.