Genetic Diversity and Fine-Scale Genetic Structure in Syagrus oleracea (Mart.) Becc.: A Survivor Palm Species of the Semiarid

The species Syagrus oleracea is a native palm tree, present in highly impacted environments of the Brazilian semiarid. This species is exploited for use in the manufacture of folk crafts and for human and animal feeding. The objective of this study was to characterize the diversity and genetic structure of individuals from different populations, located in anthropic regions of the Brazilian semiarid region, in the state of Minas Gerais. This information is important for species management and conservation strategies. Thus, we used ISSR molecular markers in 157 individuals in seven populations of S. oleracea. Looking at the results, one can observe that amplifications of 109 locos occurred and that an average of 18, 17 alleles were found on each primer, values that demonstrated that the ISSR technique used was highly efficient. The genetic diversity indexes were observed, showing expected heterozygosity (He) and Shannon index (I) of 0.260 and 0.383, respectively. The number of alleles observed (na) among populations was 1.954 and the effective number of alleles (ne) was 1,606. Individuals of the populations are genetically structured in groups (demes) in a random and aggregate manner. We obtained the amplification of 109 ISSR loci, with a mean of 18.2 alleles per primer. The genetic diversity indexes revealed expected heterozygosity (He) and Shannon index (I) of 0.260 and 0.383, respectively. The number of alleles observed (na) was 1.954 and the effective number of alleles (ne) was 1.606. The value of the indirect gene flow found was 1.546. Evidence of high levels of kinship in populations was found, possibly due to low migration rates and geographical barrier. In addition, we observed signs of overexploitation in the areas, which consequently leads to a low rate of gene flow, occurring inbreeding among the sampled populations of Syagrus oleracea.


Introduction
The species Syagrus oleracea (popularly known as guariroba and catolé) is a palm tree of the family Arecaceae, native to Brazil, of a long size that can reach more than 20 m in height, a olitary trunk, they maintain their leaves throughout the year organized as a spiral and a little arched (Lorenzi et al., 2004).It has an abundance of fruits, presents on average 70 fruits, which germinate generally in 2-3 months (Lorenzi et al., 2004).The fruits have fibrous pulp, the almonds are solid, hard, white in color and quite oily (Santelli, 2006).S. oleracea, like most palm species, often has low percentages of germination, loses viability when they become dehydrated and thereby reduce germination (Martins et al., 1999).
The palm heart with bitter flavor is one of the characteristics of this species.The peroxidase and polyphenoloxidase enzymes act on the phenolic compounds (Shimokomaki et al., 1975), and render the pH value of palmitic acid (around 5.7) (Carneiro et al., 2003).The palm heart of the species becomes a delicacy, valuable and has great acceptance among the consumers (Jaime et al., 2007).
The S. orelarea palm is found in the states of São Paulo, Minas Gerais, Mato Grosso, Goiás, Mato Grosso do Sul and Bahia (Lorenzi, 1992).Although most palm trees are not yet considered endangered species, the disordered use by human action such as fragmentation decreases both the amount of subpopulation elements and pollinators, which consequently reduces the range of gene flow between subpopulations making them vulnerable.
Therefore, knowing the distribution patterns of genetic variation within and between natural populations is necessary for the establishment of conservationist and sustainable use practices (Frankel, 1983).The molecular markers are widely used to describe patterns of genetic variability in natural plant populations.These tools make it possible to detect variations in the genome at any stage of plant development (Kamada et al., 2009).
The Inter Simple Sequence Repeats (ISSR) molecular markers stand out in genetic analyzes, make it possible to verify intra and intertaxonomic variability in phylogenetic reconstructions, at species levels or above, and also through the molecular markers make it possible to obtain high degree of polymorphism, reproducibility and because they are considered low cost (Salimath et al., 1995).
In this context, the objective of this study was to characterize the genetic diversity, spatial genetic structure of Syagrus oleracea by ISSR molecular markers in natural populations of the Brazilian semiarid.

Study Area
Samples were obtained from populations located in northern Minas Gerais-Brazil (Table 1).

Molecular Analysis
Leaves and roots of adult plants were collected from 157 individuals sampled in seven populations of Syagrus oleracea.UTM coordinates with Global Positioning System (GPS) mapped the individuals of S. oleracea.
The CTAB (cetyl trimethyl ammonium bromide) protocol proposed by Doyle and Doyle (1987) was used to extract the DNA from the leaves of each sample.
The size of the amplified fragments was estimated by comparison with a molecular marker of 100 bp (DNA Ladder).Electrophoretic separation was performed for 3 hours at 100 volts.At the end of the run, the gels were visualized in transilluminator and photodocumented.
Subsequently, the fragments were visualized and characterized according to the presence (1) or absence (0) of the fragments.Only fragments with high intensity were considered, and fragments with low reproducibility were discarded from the analyzes.A binary matrix was constructed for analyzes of diversity and genetic structure.

Analysis of the Data
Analysis of the genetic diversity of the populations was performed using the software POPGENE, V. 1.31 (Yeh, Yang, & Boyle, 1999).The number of alleles observed (na), the effective number of alleles (ne) (Kimura & Crow, 1964), the expected heterozygosity (He) (Nei, 1973), the Shannon index (I) (Lewontin, 1972), the polymorphic loci (P) and the percentage of polymorphic loci (P%).
We calculated the value of PIC (Polymorphic Information Content) that classifies the primers according to the polymorphism detection efficiency, in which values greater than 0.5 are considered quite informative, values between 0.25 and 0.50 are highly informative and lower values to 0.25 little informative (Botstein et al., 1980).
The distributions of genetic diversity among and within populations were evaluated by total heterozygosity (Ht), heterozygosity within the populations (Hs), population differentiation coefficient G ST (NEI, 1973) and gene flow (Nm) estimated by formula Nm = 0.25 (1 -G ST )/G ST (McDermott & McDonald, 1993).
For analysis of significance of the components of the variance 1,000 permutations were tested.To test the correlation between genetic identity and geographic distance between populations, the genetic identity and genetic distance estimation of Nei (1978) among the populations of S. oleracea was carried out.
The genetic similarity was also calculated by the Jaccard similarity coefficient with the use of the NTSYS software (Rohlf, 2000), in which an UPGMA grouping (unweighted pair group method with arithmetic mean) was generated.
The analysis of the fine-scale genetic structure within each population was estimated with the value of coancestria using the kinship coefficient between the pairs of individuals for the distance classes (Hardy, 2003).We used the program Spagedi, version 1.2 (Hardy & Vekemans, 2002), in the estimations of coefficient of kinship as coefficient consideration equals zero for inbreeding.For half-time jackknife re-sampling, confidence intervals were constructed based on the standard error of the mean of the estimates obtained.The confidence intervals have a 95% probability of the average co-estimate coefficient estimated for each distance class (Hardy & Vekemans, 2002).

Genetic Diversity
The amplification of the six selected ISSR primers revealed 109 fragments, which ranged from 14 to 22 per primer and average of 18 fragments.The mean value of PIC for all primers was 0.344, with a highest value found of 0.377 and the lowest value of 0.292.The sizes of the fragments obtained ranged from 200 to 2300 nucleotide pairs.The results showed that the six primers used were efficient for the detection of polymorphism in the populations of Syagrus oleracea (Table 2).
In addition, the results revealed high polymorphism in all S. oleracea populations sampled in the different studied areas.Rossi et al. (2014) found a similar result (78.3%) in the species Mauritia flexuosa (Arecaceae).Santos (2017) studied the species Copernicia prunifera (Arecaceae), found polymorphism of 99.09%.The heterozygosity is one of the most important and most used methods to estimate genetic variability (Brown et al., 2007).It is also considered the least sensitive method to variations in sample size when compared to other measures.The average heterozygosity values expected for entomophilous cross-pollinating species is 0.167, widely dispersed species of 0.202, tropical with 0.148 and arboreal species of 0.177 (Hamrick & Godt, 1990).The values found for S. oleracea are in agreement with these authors, being considered above the average of these.
The number of alleles observed (na) among populations was 1.954, the effective number of alleles (ne) 1.606, showing no significant differences.Similar results were observed in a study developed by Vieira et al. (2015) with the species Copernicia prunifera, in which the number of alleles observed (na) 2.00 and effective alleles (ne) 1.46 were obtained.
It can be observed that in all populations the values obtained are in agreement with the average expected by the indexes indicated, and also it is found against a large part of the literature searched for palm trees with ISSR markers.The results in this study indicate the occurrence of genetic diversity, although apparently there are factors that lead to endogamy in the populations studied.

Genetic Structure
The genetic diversity based on the Hardy-Weinberg equilibrium observed among the populations was 0.244.The total heterozygosity (Ht) value among the populations was 0.345, the heterozygosity within the populations (Hs) of 0.260 and the gene flow (Nm) obtained between the populations was 1.546 (Table 3).In most cases, allogamous species present higher values of genetic diversity within populations, in relation to autogamous species (Hu et al., 2010).What was evidenced in this study, the found value of the population differentiation coefficient (G ST ) 0.244 and the heterozygosity within the populations (Hs) 0.260, showed that the genetic divergence value among the populations is lower, than the value within populations.According to Hamrick (1991), factors such as geographic barriers, ecological differences and historical processes, and the dispersal capacity of species are determinant in the population genetic structure of a species.In addition, in tree species, when populations become isolated, they cause changes in basic evolutionary processes, such as genetic drift, selection, migration and breeding system, resulting in changes in genetic diversity (Silva, 2014).
It was observed in the populations of S. oleracea estimated gene flow (Nm) of 1.546.The value found indicates low gene flow, possibly due to low seed dispersion efficiency, or the existence of a geographical barrier between the populations considered in this study.The genetic identity values of Nei (1978) among the populations studied ranged from 0.791 in Rio Pardo de Minas and Mirabela to 0.941 in Novo Horizonte and São João da Ponte.It was observed lower values in the analyzes of genetic distance estimation in Novorizonte and São João da Ponte with a value of 0.06 and the highest value in Rio Pardo de Minas and Mirabela with a value of 0.234 (Table 5).
The dendrogram generated from the genetic identity matrix allowed the observation of all populations surveyed (Figure 1), showing a range of similarity among populations from 83% to 95%.Retrating more than 87% of similarity.By the analysis of the dendrogram, we can notice the formation of two large groups: a group formed by the population of Rio Pardo de Minas and another composed by the other populations: Novorizonte, São João da Ponte, Varzelândia, Josenópolis, Japonvar and Mirabela.In addition, genetic differentiation was observed among populations, and some clusters could be visualized, that is, that there are genetic material exchanges.The results also demonstrate that some populations may require conservation strategies because they have lower genetic diversity values.Reis et al. (2017)  propose that other studies should be carried out to enable the maintenance of genetic variability and the establishment of strategies for the conservation and sustainable use of the species Syagrus oleracea.

Table 1 .
Sampling of S. oleracea populations and characterization of the areas