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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 213-221, January-December 2022 (Published Mar. 30, 2022)

Diversity and genetic structure of natural populations

of the palm tree Euterpe precatoria (Arecaceae)

Jakeline Santos Cochev da Cruz1; https://orcid.org/0000-0003-0223-4997

Juliana de Freitas Encinas Dardengo2; https://orcid.org/0000-0002-2086-4514

Alex Souza Rodrigues3; https://orcid.org/0000-0003-4040-5654

Auana Vicente Tiago2; https://orcid.org/0000-0001-9556-9491

Eliane Cristina Moreno de Pedri2; https://orcid.org/0000-0002-7044-581X

Kelli Évelin Müller Zortéa2; https://orcid.org/0000-0003-0545-6130

Magali Gonçalves Garcia4; https://orcid.org/0000-0002-3166-2483

Sandra Mara Alves da Silva Neves5; https://orcid.org/0000-0002-2065-244X

Ana Aparecida Bandini Rossi2*; https://orcid.org/0000-0002-8318-5375

1. Education Department of Mato Grosso State (SEDUC-MT), Alta Floresta, Brazil; jackcochev@gmail.com

2. Mato Grosso State University, Alta Floresta, Brazil; anabanrossi@unemat.br (*Correspondence),

auana_bio@hotmail.com, elicmbio@gmail.com, kelli.zortea@unemat.br

3. North Fluminense State University, Campos dos Goytacases, Brazil; alexsouzarodrigues@outlook.com

4. Federal University of Pará, Altamira, Brazil; mgarcia.bio@gmail.com

5. Mato Grosso State University, Cáceres, Brazil; ssneves@unemat.br

Received 08-X-2021. Corrected 07-I-2022. Accepted 22-III-2022.

ABSTRACT

Introduction: The natural ecosystems of northern Mato Grosso, Brazil, are in process of fragmentation, mainly

due to population growth and the expansion of agriculture. This endangers the palm Euterpe precatoria (locally

known as açaí), used for construction, palm hearts, juices and ice cream.

Objective: To evaluate the local diversity and genetic structure in native populations of E. precatoria.

Methods: We collected leaves from 106 fruiting palms from five populations in Mato Grosso State, for analysis

of microsatellite markers with Polymerase Chain Reaction.

Results: The five SSR loci revealed a total of 30 alleles, ranging from 5 (EE23 and EE43) to 7 (EE2 and EE15),

with an average of 6 alleles per locus. The mean PIC was 0.74 and confirmed low heterozygosity and inbreeding.

The UPGMA dendrogram produced two groups and molecular variance revealed greater genetic differentiation

within populations. The high levels of homozygous microsatellite loci indicate low genetic diversity.

Conclusions: These populations have low gene diversity, high average number of alleles per locus, and rare and

exclusive alleles. We recommend the establishment of permanent conservation units with corridors among them.

Key words: acai; microsatellite; genetic resource; Amazon; conservation.

https://doi.org/10.15517/rev.biol.trop..v70i1.42942

GENÉTICA

The natural ecosystems of the northern

region of Mato Grosso (Brazil) are in pro-

cess of fragmentation, mainly due to popula-

tion growth and the expansion of agricultural

frontier. As a consequence, the formation of

mosaics of remaining vegetation is increas-

ing, reducing the size of the populations and

causing changes in the ecological and genetic

214 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 213-221, January-December 2022 (Published Mar. 30, 2022)

processes of the natural species that occur there

(Dardengo et al., 2021).

This exploitation contributes to the deg-

radation of the environment and has become

a worrying factor for the preservation of the

species Euterpe precatoria Mart. (açaí). This

species occurs in an Amazonian environment

and has high commercial interest, being used

by Amazonian people for civil construction and

for food, both the fruit for juices and ice cream,

and the heart of palm (Arruda et al., 2014).

The illegal logging of adult individuals of

E. precatoria to remove the heart of palm and

wood, causes a reduction in the number of indi-

viduals, thus, alleles of interest are lost and not

transmitted to the next generations (Azêvedo et

al., 2017). Knowing the distribution of genetic

diversity among and within natural popula-

tions is very important for the development of

conservation strategies. In addition, it allows

a better understanding of how the selection is

working, therefore, a widely used way to detect

this variability is through studies of the genetic

structure of natural populations (Estopa et al.,

2006). Genetic studies with native plant species

must be carried out to gather information that

contributes to in situ conservation, sustain-

able management and the formation of seed

collection areas, in order to recover degraded

areas (Pinto et al., 2009). Molecular markers

have been used frequently in studies like these

(Ramos et al., 2021).

Among the molecular markers used in

studies about forest fragmentation, microsatel-

lites or Simple Sequence Repeat-SSR stand

out, which are codominant and specific to each

species, with possible transferability to other

species of the same genus (Faleiro, 2007).

SSRs are markers widely used to estimate

genetic parameters of populations, gene flow

patterns and kinship, being abundant and well

distributed across the plant genome. According

to Manel et al. (2003), the use of molecular

data in studies of population and landscape

genetics, contributes to the analysis of the gene

flow rate, genotype distribution, genetic adap-

tation, adaptive differentiation and speciation.

Thus, we aim to assess the diversity and genetic

structure of native populations of E. precatoria,

located in micro-region II-North, State of Mato

Grosso, Brazil, to support strategies for manag-

ing the genetic resources of the species for its

conservation or domestication.

MATERIALS AND METHODS

Study area and Sampling: The study

was carried out in five municipalities (Alta

Floresta, Carlinda, Nova Bandeirantes, Nova

Monte Verde and Paranaíta) that compose the

planning micro-region II-North of the state

of Mato Grosso, Brazil. This area is a natural

landscape that once was a unique forest, and it

was selected for the study because there is a lot

of deforestation pressure.

For the analysis of genetic diversity and

structure, leaf material was collected from

106 adult individuals (individuals in phase of

fructification) of E. precatoria distributed in

five populations located in the municipalities.

Sampling was carried out randomly in the for-

est fragment, each individual sampled was an

average of fifty meters apart.

DNA extraction and Polymerase chain

reaction (PCR) amplification: The total

genomic DNA was extracted from approxi-

mately 100 mg of leaves, using the CTAB

method described by Doyle and Doyle (1990),

with modifications suggested by Soares et al.

(2016) and Zortéa et al. (2016). The DNA con-

centration were estimated by comparison with

the lambda DNA and the samples were diluted

to a concentration of 15 ng.μL-1 to perform

the amplifications.

Seven isolated microsatellite primers

(SSR) that were characterized by Gaiotto et al.

(2001) were tested in an initial PCR amplifica-

tion using three E. precatoria individuals. Of

the seven SSR primers tested, five amplified

the E. precatoria genome and were selected for

genetic diversity analysis.

The amplification reactions via poly-

merase chain reaction (PCR) were performed

with a final volume of 13 μL: 0.12 μL of Taq

DNA polymerase (5 U / μL); 1.5 μL 10x Buffer

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[10 mM Tris-HCl (pH 8.3); 50 mM KCl; 0.1

% tween; 10 mM MgCl2]; 1.04 μL MgCl2

(25 mM); 2 μL of primer R and F (2 μM); 1.5

μL of dNTPs (1 mM); 2 μL of DNA (30 ng);

completing the volume with ultrapure Milli-Q

water autoclaved.

The amplifications were carried out in a

Biocycler thermocycler under the following

conditions: an initial denaturation cycle at 94

ºC for 15 minutes, followed by 35 cycles: a)

denaturation at 94 ºC for thirty seconds; b)

annealing at 58-63 °C (depending on the primer

used) for 1 minute and 30 seconds; c) exten-

sion of 72 ºC for 1 minute and a final extension

cycle of 72 ºC for 7 minutes (Aguilar-Barajas

et al., 2014).

The amplification products were separated

by electrophoresis on 3 % agarose gel (mv-1)

in running buffer TBE 1X (EDTA 0.02 M;

boric acid 0.89 M; Tris-base 0.89 M), in con-

stant voltage of 70 V for approximately four

hours, to avoid overlap of the amplification

products. The fragments were scored using

GelQuantPRo®.

Data analysis: We used the Power Marker

program (Liu & Muse, 2005) to assess allelic

frequency, genetic diversity, the observed and

expected heterozygosity, fixation index and the

polymorphism information content (PIC). Nei

(1983) matrix of genetic distance between E.

pracatoria trees was estimated using the same

program. This matrix was imported by MEGA

3.1 to construct a dendrogram of mean distance

using the unweighted pair group method with

arithmetic mean (UPGMA). The presence of

null alleles was check by the use of Micro-

Cheker program (Van Oosterhout et al., 2004).

The Structure program (Pritchard et al.,

2000), which is based on Bayesian statistics,

was used to indicate the number of genetic

groups (K). We conducted 20 runs for each K

value, with 250 000 burn-ins and 500 000 Mar-

kov chain Monte Carlo simulations. To deter-

mine the most probable value of K, we used

the criteria proposed by Evanno et al. (2005).

The most probable number of different

populations that contributed to the genetic

composition of the E. precatoria populations

under study was verified by the ΔK estimate

described by Evanno et al. (2005). The ΔK

was estimated for K ranging from one to eight,

with the highest value occurring for the number

of genetically distinct populations that best

explains the data set.

Principal coordinate analysis (PCoA),

deviations of the Hardy-Weinberg equilibrium,

analysis of molecular variance (AMOVA) and

the analysis of rare (AR) and exclusive alleles

(EA) were performed using the GenAlEx 6.5

program (Peakall & Smouse, 2006).

RESULTS

Populational Genetic Diversity of Euter-

pe precatoria: The microsatellite loci showed

a total of 30 alleles in E. precatoria, ranging

from 5 to 7 alleles, with an average of 6 alleles

per locus. The content of polymorphic informa-

tion (PIC) was above 0.60 for all loci (Table 1).

The average expected heterozygosity (He) was

higher than the expected heterozygosity (Ho),

with a higher frequency of homozygotes in the

sampled E. precatoria populations.

The fixation index had an average of 0.77

and all loci showed positive fixation index,

with locus EE43 presenting f = 1, in other

words, this locus did not reveal any heterozy-

gote in the populations (Table 1), confirming

the low heterozygosity and the high inbreeding

in the studied E. precatoria populations.

Nei’s genetic distance (Nei, 1972) revealed

that the NMV and AF populations are the most

dissimilar, that is, the most genetically differ-

ent, being geographically distant at approxi-

mately 105 km. The PR and NMV populations

showed the greatest genetic similarity and were

approximately 87 km apart, indicating a greater

possibility of flow between the sampled popu-

lations (Table 2).

Table 3 shows the results of genetic diver-

sity by population. The PR population was the

one with the highest number of alleles, fol-

lowed by the populations of NMV and NB. The

analysis by loci and the analysis by popula-

tions showed that the number of homozygotes

216 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 213-221, January-December 2022 (Published Mar. 30, 2022)

was greater than that of heterozygotes. The

fixation index was positive for all popula-

tions, which confirms the greater number of

homozygotes and the low frequency of hetero-

zygotes in the sampled populations. The AF

population showed the lowest He (0.441) and

f (0.537) values and the highest heterozygosity

observed. It was not observed any null allele in

the analysis.

Genetic structure and populational dif-

ferentiation of E. precatoria: The dendro-

gram generated from the genetic distance of

Nei (1983) by the UPGMA method formed

two groups. Group 1 (G1) was formed by the

populations of Alta Floresta (AF) and Carlinda

(CR), while in group 2 (G2) the populations of

Nova Bandeirantes (NB), Nova Monte Verde

(NMV) and Paranaíta (PR) were allocated. In

TABLE 2

Geographical distance (above the diagonal) in kilometers (km) and Nei’s Genetic Distance (1983) (below the diagonal)

among the populations of Euterpe precatoria sampled in the microregion II-Norte, in the State of Mato Grosso, Brazil

Populations AF CR NB NMV PR

AF - 73.044 150.557 104.999 86.811

CR 0.584 - 213.678 166.383 110.077

NB 0.2778 0.246 - 48.6 127.97

NMV 0.060 0.440 0.445 - 86.811

PR 0.243 0.381 0.605 0.706 -

AF: Alta Floresta; CR: Carlinda; NB: Nova Bandeirantes; NMV: Nova Monte Verde; PR: Paranaíta.

TABLE 3

Estimation of the genetic diversity parameters of five populations of Euterpe precatoria naturally occurring

in the micro-region II-North, State of Mato Grosso, Brazil

Population N A HeHof

AF 21 15 0.441 0.207 0.537

CR 19 15 0.564 0.200 0.652

NB 23 17 0.626 0.156 0.754

NMV 20 19 0.563 0.180 0.686

PR 23 21 0.598 0.130 0.785

Average 21.2 17.4 0.559 0.175 0.692

AF: Alta Floresta; CR: Carlinda; NB: Nova Bandeirantes; NMV: Nova Monte Verde; PR: Paranaita N = Number of

individuals; A = Allelic richness; He = expected heterozygosity; Ho = observed heterozygosity; f = Intrapopulational fixation

index.

TABLE 1

Genetic parameters for five microsatellite loci, based on the amplification of the DNA of 106 Euterpe precatoria

individuals from five populations sampled in the micro-region II-North in the State of Mato Grosso, Brazil

Loci Na He Ho f PIC

EE2 07 0.73 0.06 0.91* 0.70

EE15 07 0.83 0.05 0.94* 0.80

EE23 05 0.76 0.11 0.85* 0.72

EE43 05 0.70 0.00 1.00* 0.65

EE54 06 0.82 0.64 0.22* 0.79

Total 30 - - - -

Average 06 0.76 0.18 0.77* 0.74

Number of alleles (Na), Expected heterozygosity (He), Observed heterozygosity (Ho), Fixation index (f) and Polymorphic

Information Content (PIC). *P < 0.05.

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Fig. 1. A. Geographical location of the five populations of Euterpe precatoria under study in micro-region II-North in

the State of Mato Grosso, Brazil. B. Dendrogram obtained by the UPGMA method, based on the genetic distances of Nei

(1983). Branch reliability was tested by bootstrap analysis using 1 000 replications. C. Grouping of 106 individuals from

five populations of Euterpe precatoria based on five SSR loci using the “Structure” Program. Individuals are represented

by vertical columns and are shaded according to their group (two genetic groups, K = 2). PAR: Paranaíta; NB: Nova

Bandeirantes; NMV: Nova Monte Verde; CAR: Carlinda and AF: Alta Floresta.

218 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 213-221, January-December 2022 (Published Mar. 30, 2022)

G2, two subgroups are formed, the first sub-

group formed by the population of NB and a

second subgroup formed by the populations

of NVM and PR, the geographic distance may

have been the factor that contributed to the

similarity between these populations, since

they are neighboring municipalities (Fig. 1A,

Fig. 1B).

The Bayesian analysis performed by the

“Structure” program corroborates the result

obtained by the UPGMA method, with the

formation of two distinct groups (k = 2). The

two genetic groups found in the evaluated

sample are represented in black (group 1) and

gray (group 2), indicating that the populations

of PR, NB and NMV are constituted mainly by

the genetic group 1, while the populations of

AF and CR by group 2 (Fig. 1C).

The PCoA explained 29.79 % of the total

variation, with 18.06 % for the first component,

11.73 % for the second (Fig. 2).

As in the other clusters, we observe the

formation of two groups and the individuals

were allocated according to the previous group-

ings generated by UPGMA (Fig. 1B) and by

Structure (Fig. 1C). AMOVA revealed that 73

% of the total variance occurred within popula-

tions and 27 % among populations (Fig. 3).

DISCUSSION

In the work of Oliveira et al. (2010), with

E. oleracea, 42 alleles were found for the set

of five primers used, ranging from 10 alleles

(EE23) to 04 alleles (EE2), values higher than

those found in this study, while Azêvedo et al.

Fig. 2. Graphic dispersion from the analysis of the main coordinates of 106 individuals of Euterpe precatoria from sampled

populations in microregion II-Norte, State of Mato Grosso, Brazil. AF: Alta Floresta; CR: Carlinda; NB: Nova Bandeirantes;

NMV: Nova Monte Verde; PR: Paranaita. Coord.: Coordinate.

Fig. 3. Analysis of molecular variance (AMOVA) of the

four populations of Euterpe precatoria studied based on

five SSR markers.

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(2017) found 08 alleles, with an average of 1.6

alleles per locus, for E. precatoria, a number

lower than that found in this study, that is, the

data found in this study are within the expected

for the species.

All loci analyzed in this study were highly

informative, confirming the transferability of

the microsatellites from E. edullis to E. preca-

toria. According to Botstein et al. (1980) Poly-

morphism Information Content (PIC) values

below 0.25 are considered uninformative, those

that reveal values between 0.25 and 0.50 are

classified as moderately informative and above

0.50 highly informative. Oliveira et al. (2010)

when transferring the same markers from E.

edullis to E. oleracea found the PIC value of

0.86 for primers EE23 and EE54, highly infor-

mative values.

In this study, all loci presented f values

above 0.22, indicating excess of homozygotes

in the populations, according to Azevedo et

al. (2007), the fixation index value varies

between -1 and +1, positive values indicate

excess homozygote, negative values excess of

heterozygotes and zero is the value found for

a population in Hardy-Weinberg equilibrium.

Among the results found, this study high-

lights the low heterozygosity and the high

rate of inbreeding in all loci, which can be

influenced by the low flow of dispersers and

also by the reducing number of E. precatoria

individuals in the fragments. Another explana-

tion for the high inbreeding values lies on the

presence of null alleles, because it increases

the number of homozygous individuals, since

just one of the alleles amplifies itself in cases

of null allele in heterozygous plant (Nybom,

2004), however, it was not found any null allele

in the analysis.

The forest fragmentation found in the field

can be one of the main factors for the high rate

of inbreeding, since fragmentation becomes

an “obstacle” for the migratory flow of animal

species that provide systemic services to the

plant, such as dispersers. The E. precatoria

fruits are part of the diet of several animals

that are seed dispersers in the forest, the main

wild animals that help in the dispersion of the

species are birds of the Psittacideae, Rampha-

stidae and Cracidae families (Rocha & Viana,

2004). Another factor that may also have

contributed to inbreeding is the species life

strategy, which forms a seedling bank close to

the mother plant (Oliveira et al., 2010; Ramos

et al., 2021), in the other hand, studying the

same species, have found a high expected het-

erozygosity, which according to the authors, is

explained by the allogamy. So once again, we

can say that the fragmentation may be the cause

for this increase of homozygous levels.

Inspite, of the low number of sampled

individuals, the presence of rare (PR, NMV and

CR population) and exclusive alleles (NMV

population), indicated the importance of these

populations, thus, they need preservation strat-

egies for specimen conservation. Dias Filho

(2006) in a study with the species Eutepe edul-

lis identified seven exclusive alleles in a natural

population in the Municipal Reserve of Santa

Genebra, São Paulo State, using the same set

of microsatellites tested in this work and used

this information to justify the conservation of

the species in the area.

For this study, the K value indicated the

presence of two distinct genetic groups, or

populations, among the individuals studied,

showing that even with the geographical dis-

tance and fragmentation in the landscape, there

is still a connection among the populations,

because there are some corridors of vegetation

among them.

The values presented for E. precatoria in

this study, indicated the existence of greater

variability within populations, a common fact

for allogamous species. Oliveira et al. (2010)

also identified high genetic differentiation

(69.88 %) within natural populations of E.

oleracea, corroborating the values obtained for

natural populations studied in this work.

The populations studied present levels of

gene diversity, high average number of alleles

per locus and presence of rare and exclu-

sive alleles. It is also important to infer that

the development of agricultural activities that

has been occurring in an accelerated manner

around the forest fragments will provide an

220 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 213-221, January-December 2022 (Published Mar. 30, 2022)

increase in the isolation of these populations

and loss of these alleles, so the establishment

of permanent conservation units with corridors

among them, could be a valuable tool to pre-

serve genetic diversity among the individuals

of these natural populations. We strongly sug-

gest more studies with the species, with more

loci and individuals sampled on it.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

We thank the Fundação de Amparo à Pes-

quisa do Estado de Mato Grosso-FAPEMAT

for granting a doctoral scholarship to the first

author during the period from October 2015 to

March 2017 and for funding the research proj-

ect: “Landscape dynamics of forest fragments

and structure population genetics of two native

Amazonian species”, Process nº 229312/2015.

RESUMEN

Diversidad y estructura genética de poblaciones

naturales de la palmera Euterpe precatoria (Arecaceae)

Introducción: Los ecosistemas naturales del norte de

Mato Grosso, Brasil, están en proceso de fragmentación,

principalmente debido al crecimiento de la población y la

expansión de la agricultura. Esto pone en peligro la palma

Euterpe precatoria (localmente conocida como açaí), utili-

zada para la construcción, extracción de palmito, prepara-

ción de jugos y helados.

Objetivo: Evaluar la diversidad local y estructura genética

en poblaciones nativas de E. precatoria.

Métodos: Recolectamos hojas de 106 palmas fructíferas

de cinco poblaciones en el estado de Mato Grosso, para

análisis de marcadores microsatélites con el método de

Reacción en Cadena de la Polimerasa (PCR).

Resultados: Los cinco loci SSR revelaron un total de

30 alelos, que van desde 5 (EE23 y EE43) hasta 7 (EE2

y EE15), con un promedio de 6 alelos por locus. El PIC

medio fue de 0.74 y confirmó baja heterocigosidad y endo-

gamia en las poblaciones. El dendrograma UPGMA pro-

dujo dos grupos y la varianza molecular reveló una mayor

diferenciación genética dentro de las poblaciones. Los loci

de microsatélites presentaron un alto nivel de homocigotos

lo que indica una baja diversidad genética.

Conclusiones: Estas poblaciones tienen baja diversidad

genética, alto promedio de alelos por locus y alelos raros y

únicos. Recomendamos el establecimiento de unidades de

conservación permanentes con corredores entre ellas.

Palabras clave: acai; microsatélite; recurso genético;

Amazonas; conservación.

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