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Spondias mombin (Anarcadiaceae):

molecular characterization and conservation

Kelli Évelin Müller Zortéa

,2,3

; https://orcid.org/0000-0003-0545-6130

Ana Aparecida Bandini Rossi

1,2,3

; https://orcid.org/0000-0002-8318-5375

Auana Vicente Tiago

1,3

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

Elisa dos Santos Cardoso

1,3

; https://orcid.org/0000-0001-9477-1743

Joyce Mendes Andrade Pinto

; https://orcid.org/0000-0002-9484-1868

Eulalia Soler Sobreira Hoogerheide

; https://orcid.org/0000-0003-0944-3898

1. Programa de Pós-Graduação em Biodiversidade e Biotecnologia (Rede Bionorte), Universidade do Estado de

Mato Grosso - Carlos Alberto Reyes Maldonado, Alta Floresta, Mato Grosso, Brasil; kellimullerz@gmail.com

(Correspondence*), anabanrossi@unemat.br, auanavt@gmail.com, elisabyo@gmail.com

2. Programa de Pós-Graduação em Genética e Melhoramento de Plantas, Universidade do Estado de Mato Grosso -

Carlos Alberto Reyes Maldonado, Alta Floresta, Mato Grosso, Brasil.

3. Laboratório de Genética Vegetal e Biologia Molecular, Universidade do Estado de Mato Grosso - Carlos Alberto

Reyes Maldonado, Alta Floresta, Mato Grosso, Brasil.

4. Embrapa Agrossilvipastoril, Sinop, Mato Grosso, Brasil; joyce.andrade@embrapa.br,

eulalia.hoogerheide@embrapa.br

Received 12-II-2021. Corrected 10-VII-2021. Accepted 01-IX-2021.

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 F

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.

Key words: conservation; genetic resources; genetic variability; microsatellite; yellow mombin.

Müller Zortéa, K. E., Bandini Rossi, A. A., Vicente Tiago,

A., dos Santos Cardoso, E., Mendes Andrade Pinto,

J., & Soler Sobreira Hoogerheide, E. (2021). Spondias

mombin (Anarcadiaceae): molecular characterization and

conservation. Revista de Biología Tropical, 69(3), 1023-

1036. https://doi.org/10.15517/rbt.v69i3.45810

https://doi.org/10.15517/rbt.v69i3.45810

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Brazil has a high potential for the produc-

tion of numerous varieties of fruits due to its

territorial extension, varied types of soils, and

climatic conditions (Carvalho et al., 2017).

It also has a great diversity of native fruits,

many of them still unknown or little exploited

commercially, but that have increasing market

value, among which is the yellow mombin

(Spondias mombin L.) (Freitas & Mattietto,

2013). This tree species belongs to the family

Anacardiaceae and its fruit stands out for its

sensory and functional qualities (Pinto et al.,

2003). In addition, the fruit has high levels of

carotenoids, tannins and phenolic compounds,

and is considered a natural source of antioxi-

dants (Freitas & Mattietto, 2013; Mattietto et

al., 2010). S. mombin is among the Brazilian

species that has the greatest economic poten-

tial (Lederman et al., 2008); however, it is in

a domestication phase, and information that

would assist in the deployment of viable com-

mercial orchards is scarce (Souza et al., 2006).

The type of exploitation, the lack of commer-

cial crops and the high loss of fruits during

harvest and transport cause annual variations

in the harvests, which directly influence its

industrialization and functioning of the fruit

processing industries (Santana, 2010).

The domestication and incorporation of

native species into productive systems require

information on genetic variation and knowl-

edge of the size and distribution of genetic

variability in natural populations (Costa et al.,

2011). In addition, knowledge of the genetic

structure of populations and their levels of

genetic diversity are important in order to

understand how selection acts as a function of

adaptability and how the effects of environ-

mental fragmentation may influence population

dynamics, thus helping to guide more efficient

breeding and conservation programs (Estopa et

al., 2006; Sujii et al., 2015).

Genetic diversity can be verified through

morphological, agronomic, molecular charac-

teristics, among others (Dardengo et al., 2021;

Giles et al., 2018; Giustina et al., 2017; Santos

et al., 2020). However, molecular techniques

make it possible to accelerate the process of

analysis of variability and selection especially

when working with perennial species, since

they do not require the plant to complete its

reproductive cycle, do not suffer interference

from the environment and, in addition, have

high efficiency for material discrimination

(Ferreira & Grattapaglia, 1998).

Recently, some studies have contributed

to the characterization of the Spondias genus

through molecular phylogeny (Silva et al.,

2015; Machado et al., 2015), and much infor-

mation is now available in GenBank. For S.

mombin, the assembly of the complete chloro-

plast genome (Santos & Almeida, 2019) and its

mitochondria (Martins et al., 2019) has already

been obtained. The genetic diversity of S. mom-

bin has been evaluated by means of isoenzymes

(Gois et al., 2009), morphological traits of the

fruits (Silva et al., 2017a), molecular markers,

RAPD (Lima et al., 2011) and ISSR (Silva et

al., 2017b). However, no studies are available

regarding the diversity and genetic structure

based on microsatellite molecular markers.

Microsatellites are one of the most poly-

morphic classes of molecular markers available

today and have advantages over other markers

based on PCR (polymerase chain reaction)

because they are codominant, highly reproduc-

ible, require small amounts of DNA, and have

high resolution power and high levels of poly-

morphism (Caixeta et al., 2016). The greatest

limitation to the use of this type of marker is the

lack of initiators available for all species, since

the marker is specific (Arnold et al., 2002) as

well as the great amount of work required for

the development and isolation of loci contain-

ing these markers (Zane et al., 2002). How-

ever, the flanking regions of microsatellites are

generally conserved between nearby species

or genera (Arnold et al., 2002; Caixeta et al.,

2016), thus enabling the transferability of these

markers between some species. Aguilar-Barajas

et al. (2014) were able to select 14 polymorphic

microsatellites for Spondias radlkoferi Donn.

Sm., of which 12 were successfully transferred

to S. mombin, which enabled its use in diversity

studies of this species.

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Due to an increase in the demand for the

fruits of S. mombin and the lack of knowledge

that would assist in the implementation of com-

mercial plantations in the future, especially

with regard to understanding of the genetic

diversity that the species possesses, this study

aimed to evaluate, by means of microsatellite

molecular markers, the diversity and genetic

structure of natural populations of S. mombin,

in the state of Mato Grosso, Brazil.

MATERIALS AND METHODS

Study area: The study was conducted in

10 municipalities in the state of Mato Grosso.

In each municipality, an area in which the

species naturally occurred was selected. Each

selected area was evaluated as a natural popula-

tion of S. mombin. We sampled 139 individuals

distributed as follows: Alta Floresta (AF) 19,

Apiacás (AP) 7, Cáceres (CC) 15, Colíder

(CL) 14, Marcelândia (MR) 18, Nobres (NB) 8,

Nova Bandeirantes (NA) 18, Porto Estrela (PE)

15, Tangará da Serra (TS) 17 and Vila Bela da

Santíssima Trindade (VB) 8. All the sampled

areas have strong anthropogenic influence,

with isolated trees in the middle of the pasture

matrix, in the urban matrix or in disconnected

forest fragments. The sampled populations of S.

mombin come from the three biomes that exist

in the state of Mato Grosso, namely the Ama-

zon, Pantanal and Cerrado biomes, according

to the delimitation of biomes proposed by the

Ministry of the Environment (2018).

The Amazon is one of the biomes with the

greatest richness of species in the world, but

it is also marked by environmental degrada-

tion. The Amazon region where sample collec-

tions were carried out has mostly an Am-type

climate, with defined dry and rainy seasons,

a mean annual temperature above 26 °C and

annual rainfall capacity of between 2 500 to 3

100 mm (Alvares et al., 2013).

The Pantanal biome is a large wetland

mainly located in Brazil. It has natural resourc-

es of great importance, but is one which has

been put at risk due to unsustainable land

use and occupation practices, such as the

conversion of natural vegetation into cultivated

areas and pastures (Bergier et al., 2018). The

region where the sample collections were car-

ried out has an Aw-type climate, with annual

precipitation ranging from 1 300 to 1 600 mm

and a mean annual temperature of 24 to 26 °C

(Alvares et al., 2013).

The Cerrado is the biome that occupies

20 % of the Brazilian territory; however, it is

extremely anthropized and much of the original

cover has been replaced by pasture areas or

temporary crops such as soybeans, maize and

rice (Duarte & Leite, 2020). The sample collec-

tion region has an Aw-type climate, with annual

precipitation ranging from 1 900 to 2 200 mm

and a mean annual temperature of 24 °C (Alva-

res et al., 2013).

Collection of plant material and DNA

extraction: Sampling of populations was car-

ried out by randomly prioritizing individuals of

reproductive age and with greater geographical

distance from each other. Leaf material was

collected from each selected individual with

a preference for young leaves, without dam-

age and/or signs of disease. Control material

was herborized and deposited in the Southern

Amazon Herbarium - HERBAM, Mato Grosso

State University, Alta Floresta, MT, according

to the methodology of Fidalgo and Bonomi

(1989) and under the registry numbers: 15 289,

15 290, 15 291, 15 292 and 15 293.

The total genomic DNA was extracted

from approximately 100 mg of the foliar mate-

rial following the CTAB method described by

Doyle and Doyle (1987), with modifications

for the species: an increase in the concentration

of Polyvinylpyrrolidone (PVP) from 1 to 2 %

and of β-mercaptoethanol from 0.2 to 1.8 %,

an addition of 0.4 % proteinase K in the extrac-

tion buffer, reduction of the incubation time in

a water bath from 60 minutes to 30 minutes,

while maintaining the temperature at 65 °C.

Genotyping: Amplifications were per-

formed via PCR using seven microsatellite

markers developed by Aguilar-Barajas et al.

(2014) for Spondias radlkoferi and with proven

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transfer to S. mombin. The reactions were per-

formed in a final volume of 10 µl, containing

1.5 µL buffer 10x (100 mM Tris-HCl pH 8.3;

500 mM KCl), 1.5 mM MgCl2, 0.2 mM of

each dNTP, 0.5 µM of each primer (F and R);

0.12 µL of Taq DNA polymerase (5 U/µL); 20

ng of DNA and ultrapure water. Amplifications

were conducted using an Aeris

thermocycler

(Esco

) following the method described by

Aguilar-Barajas et al. (2014).

The primers were marked using different

fluorochromes to allow the analysis of the PCR

products through duplex and triplex systems

in an automatic sequencer (model ABI 3 730,

Applied Biosystems). The amplified fragments

were detected using the software GeneMarker

V. 2.6.3. The fragment size information was

used to assemble the matrix with each primer

pair for each individual, which was subjected to

analysis of the diversity and genetic structure.

Data analysis: The genetic diversity of S.

mombin populations was characterized as the

total number of alleles, number of alleles per

locus (Na), observed heterozygosity (Ho) and

expected (He) in Hardy-Weinberg equilibrium

and polymorphic information content (PIC).

The inbreeding coefficient (ƒ) was also cal-

culated according to the method by Weir and

Cockerham (1984) for each population and for

each locus. These analyses were performed

with the aid of Power Marker software v. 3.25

(Liu & Muse, 2005). Alleles were classified

according to their frequency and occurrence.

As for the frequency, alleles with a frequency

equal to or less than 0.05 were considered

rare, and those that were common were those

with a frequency greater than 0.05 (Sebbenn,

2003). Regarding occurrence, alleles were con-

sidered to be widely distributed if they were

found in more than 25 % of the populations,

and of local distribution if they were found

in less than 25 % of the analyzed populations

(Sebbenn, 2003). Alleles that occurred in only

one population, regardless of frequency, were

considered exclusive. For each population,

the rare and exclusive alleles were classi-

fied. The classification proposed by Sebbenn

(2003) was used for local rare, widely distrib-

uted rare, local common and widely distributed

common alleles.

The analysis of molecular variance

(AMOVA) was used to infer the genetic struc-

ture of populations and was performed accord-

ing to Excoffier et al. (1992) with the aid

of Arlequin 3.01 software (Excoffier et al.,

2006). The gene flow (Nm) was obtained in

the PopGene program 1.32 (Yeh et al., 1999),

via the equation: Nm = 0.25 (1 - FST)/FST.

The matrix of the genetic distance values of

Nei et al. (1983) among populations gener-

ated by the Power Marker Program v. 3.25

was imported into the GENES program (Cruz,

2016) for the construction of the dendro-

gram using the UPGMA method (Unweighted

Pair-Group Method with Arithmetic Averages).

This grouping method was chosen because it

was the one that best represented the genetic

variation under study, based on the value of

the cophenetic correlation coefficient (CCC),

stress and distortion. To verify whether there

was a correlation between genetic similarity

and geographical distance in the different pop-

ulations analyzed, a Mantel test was performed,

with 10 000 permutations, using the Genes

software (Cruz, 2016).

The Structure program (Pritchard et al.,

2000), based on Bayesian statistics, was used

to infer the number of groups (k). Twenty

runs were performed for each k value (1 to

13), 200 000 burn-ins and 500 000 Markov

chain Monte Carlo (MCMC) simulations. The

criteria proposed by Pritchard & Wen (2004)

and also the criterion proposed by Evano et al.

(2005) were used to define the most probable

K in relation to those proposed, and the results

were uploaded to the Structure Harvester site

(Earl & von Holdt, 2012).

RESULTS

Genetic diversity of S. mombin: The

seven microsatellites used from S. mombin

amplified a total of 46 alleles, ranging from 5 to

10 and averaging 6.6 alleles per locus. The PIC

ranged from 0.048 (SPO21) to 0.700 (SPO40)

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with an average of 0.317. He was higher than

Ho for six loci and presented means of 0.335

and 0.300 respectively. The fixation index

was positive and different from 0, indicating a

higher number of homozygotes in the loci and

presence of inbreeding. Only locus SPO18 pre-

sented a negative value for ƒ (Table 1).

The mean number of alleles found per

population was 18.3, among which the Cáceres

population (CC) presented the highest number

of alleles. The highest number of heterozy-

gotes, estimated by the fixation index, was

found in Nobres (NB) and the highest number

of homozygotes in the Tangará da Serra popula-

tion (TS). This result for TS is also observed by

the lower Ho value. The PE and TS populations

presented a higher rate of inbreeding compared

to the other populations (Table 2).

In the evaluation of the general allelic

frequency for the 10 populations, 10 local rare

alleles, 13 local common alleles and 23 widely

distributed common alleles were found. By

evaluating the allelic frequency by popula-

tions, we found 27 rare alleles (frequency <

0.05), distributed in 8 of the 10 populations of

S. mombin analyzed. The population Caceres

(CC) and Alta Floresta (AF) had the high-

est numbers of rare alleles (Table 3). In all,

16 unique alleles were found, that is, they

appeared in only one of the 10 populations. The

highest number of exclusive alleles was found

in the AF population (Table 3).

TABLE 1

Estimation of the genetic diversity of S. mombin obtained in seven microsatellite loci

Locus Repeat motif N Na PIC

He Ho

SPO3 (TTAA)

134 5 0.185 0.193 0.164 0.153

SPO12 (CTT)

135 10 0.250 0.256 0.178 0.310

SPO18 (CT)

126 6 0.294 0.316 0.350 -0.103

SPO21 (CT)

124 5 0.048 0.048 0.024 0.495

SPO22 (AT)

137 7 0.470 0.500 0.445 0.111

SPO31 (AAT)

136 6 0.272 0.294 0.287 0.028

SPO40 (ATGT)

99 7 0.700 0.742 0.646 0.134

Mean 127.28 6.6 0.317 0.335 0.300 0.112

N: number of individuals used in the analysis, except for missing data; Na: number of alleles; PIC: polymorphism

information content; He: expected heterozygosity; Ho: observed heterozygosity; ƒ: fixation index.

TABLE 2

Estimation of the genetic diversity of S. mombin populations obtained using seven microsatellite loci

Population N Na PIC

He Ho

AF 19 23 0.340 0.380 0.354 0.095

AP 7 17 0.368 0.486 0.833 -0.245

CC 15 24 0.326 0.359 0.349 0.073

CL 14 21 0.305 0.375 0.500 -0.076

MR 18 20 0.279 0.314 0.344 -0.064

NB 8 15 0.465 0.211 0.298 -0.351

NA 18 16 0.482 0.251 0.284 -0.091

PE 15 17 0.228 0.263 0.227 0.187

TS 17 16 0.224 0.250 0.169 0.357

VB 8 14 0.174 0.199 0.235 -0.115

Total/Species 139 46 0.317 0.335 0.300 0.112

N: number of individuals used in the analysis, except for missing data; Na: number of alleles; PIC: polymorphism

information content; He: expected heterozygosity; Ho: observed heterozygosity; ƒ: fixation index.

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Genetic structure of natural populations

of S. mombin: The analysis of molecular vari-

ance showed that there was significant genetic

differentiation (P < 0.000) among populations

and that the largest component of variation

was within populations (90.64 %) (Table 4).

The estimated gene flow was 1.99, which indi-

cates that there is no genetic isolation among

populations. The F

value (0.0963) demon-

strates that there is little genetic differentia-

tion among populations. Thus, it appears that

the gene flow maintained low differentiation

among populations.

The UPGMA dendrogram provided the

formation of two groups, the first group (GI)

formed by 8 populations and the second group

(GII) by 2 populations. The populations for

Alta Floresta (AF) and Marcelândia (MR) were

isolated in GII (Fig. 1).

The Bayesian analysis obtained by the

Structure program, grouped the 139 individu-

als of S. mombin into two groups (k = 2). The

contribution of each population to the groups

generated by the Structure program can be

verified in Fig. 2A and Fig. 2B. The popula-

tions of Alta Floresta (AF) and Marcelândia

(MR) presented greater contribution to the red

group and the rest of the populations presented

greater contribution to the green group. This

grouping is in accordance with the UPGMA

dendrogram, where the AF and MR popula-

tions are more genetically similar, and form a

TABLE 3

Rare and unique alleles found in the populations

of S. mombin for the 7 loci microsatellites

Population Rare alleles Exclusive alleles

AF 6 4

AP 0 2

CC 7 2

CL 3 3

MR 2 1

NB 2 0

NA 2 0

PE 3 3

TS 2 1

VB 0 0

Total 27 16

TABLE 4

Analysis of molecular variance (AMOVA) of the 10 populations of S. mombin studied from 7 microsatellite markers

Source of variation D.f. SS CV TV (%)

p value

Among populations 9 31.686 0.112 9.36 < 0.000

Within populations 268 257.646 1.084 90.64

Total 277 289.332 1.196

D.f.: Degrees of freedom; SS: sum of squares; CV: coefficient of variation; TV: total variation; p: are the probabilities of

having a component of variance greater than the values observed at random. The probabilities were calculated by 1 023

random permutations. F

= 0.0963.

Fig. 1. UPGMA dendrogram generated from the matrix of genetic distance by Nei et al. (1983), and based on data from

seven microsatellite loci for 10 natural populations of S. mombin. CCC = 0.78; distortion = 3.79 %; stress = 19.47 %.

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Fig. 2. Grouping analysis and geographical distribution of the groups generated by the Structure program. A. Each

population is represented by a graph, the colors indicate the proportion of contribution of each Spondias mombin population

to the groups formed in the Structure program (K = 2). B. Distribution of the two genetic groups generated by the Structure

program. The vertical lines along the X axis represent the individuals and the colored segments along the Y axis demonstrate

the coefficient of association of each individual assigned to each of the inferred K. Numbers within parentheses correspond to

populations. 1: AF (Alta Floresta); 2: AP (Apiacás); 3: CC (Cáceres); 4: CL (Colider); 5: MR (Marcelândia); 6: NB (Nobres);

7: NA (Nova Bandeirantes); 8: PE (Porto Estrela); 9: TS (Tangará da Serra); 10: VB (Vila Bela da Santíssima Trindade).

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separate group from the other populations. This

structure demonstrates that genetic variation

between populations is lower than within popu-

lations and agrees with the result of AMOVA.

The genetic structure of S. mombin is not

determined by the geographical structure, since

the most genetically similar populations are

not the closest geographically speaking. This

result is supported by the Mantel test, which

revealed a negative correlation (-0.158) and

was not significant between the matrices of

genetic similarity and geographical distance.

To better illustrate this result, one can cite the

example of the AF population that in the analy-

ses is grouped with the MR population that is

geographically further than the AP, CL and NA

populations (Fig. 2A and Table 5).

DISCUSSION

The polymorphism found in all the ana-

lyzed loci indicate the presence of genetic

diversity in the analyzed populations. The

mean number of alleles per locus found in this

study was higher than that found by Aguilar-

Barajas et al. (2014) for the same loci in 20

individuals from S. mombin in Mexico. These

authors tested the transfer of SSR primers

developed for S. radlkoferi in S. mombin and

found an average of 6.4 alleles per locus, with

a range of 5 to 7 alleles per locus. The increase

in the mean of alleles per locus in this study

may be a result of the sizable genetic differ-

ence between the individuals evaluated and

the greater geographical distance between the

areas sampled.

The expected and observed heterozygosity

were lower than that found by Aguilar-Barajas

et al. (2014) for the same loci. They were also

inferior to those found by Silva et al. (2009)

when analysis was performed by means of iso-

enzymes for the genetic diversity of four popu-

lations of S. mombin in the Rainforest Atlantic

Zone of Pernambuco, Brazil. The lower rate of

heterozygosity found for S. mombin in relation

to other tropical tree species may be related to

forest fragmentation and population size reduc-

tion. These factors can affect genetic processes

such as genetic drift, gene flow, selection,

reproduction systems, in addition to causing

reproductive isolation, increased spatial struc-

ture within the population and consequently

loss of genetic variability (Carvalho et al.,

2010; Young et al., 1996). All the populations

of S. mombin analyzed in this study were in

fragmented areas with significant anthropogen-

ic action, and some individuals were isolated in

a pasture or urban matrix.

The frequency of heterozygotes in the

population is also a measure of diversity (Nei,

1973), and a heterozygote presents variabil-

ity due to carrying two different alleles in the

TABLE 5

Genetic distance matrix* by Nei et al. (1983) and geographical distances** (Km)

among the 10 natural populations of S. mombin

AF AP CC CL MR NB NA PE TS VB

AF ---- 0.146 0.147 0.188 0.066 0.128 0.095 0.150 0.131 0.167

AP 154 ---- 0.134 0.116 0.148 0.042 0.083 0.140 0.126 0.098

CC 705 718 ---- 0.107 0.146 0.096 0.101 0.093 0.098 0.105

CL 126 253 632 ---- 0.168 0.079 0.112 0.164 0.156 0.093

MR 220 359 655 102 ---- 0.107 0.087 0.170 0.136 0.163

NB 528 570 225 432 439 ---- 0.058 0.105 0.100 0.069

NA 192 62 677 270 376 540 ---- 0.096 0.074 0.101

PE 650 674 68 570 588 157 630 ---- 0.040 0.068

TS 588 584 130 524 560 174 544 104 ---- 0.055

VB 714 680 261 685 733 403 620 298 231 ----

*Genetic distance above the diagonal. **Geographical distances (Km) below the diagonal.

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same locus. Observed heterozygosity (Ho) is

the mean frequency of heterozygous individu-

als per locus among the sample components

(Shimizu et al., 2000) and is highly influenced

by the reproductive system, with allogamous

species, such as S. mombin, generally present-

ing higher values (Faraldo et al., 2000). The

expected heterozygosity (He) is the average

frequency of heterozygotes, per locus, expected

by the Hardy-Weinberg equilibrium (Shimizu

et al., 2000) and in this case, there is no influ-

ence of the reproductive system. In the ana-

lyzed populations of S. mombin, the Ho was

higher than the He in 6 of the 10 populations

and, when Ho is higher than He, it is under-

stood that there are more heterozygotes in the

population than expected by the Hardy-Wein-

berg equilibrium (Gois et al., 2009). This result

demonstrates there is an important genetic

diversity in the populations studied.

In all, one locus and six populations had

negative fixation rates, which indicates an

excess of heterozygotes. However, the posi-

tive fixation index in most loci and in four

populations, as well as in the average for the

species, indicates the presence of inbreeding,

even if in a low proportion, due to the excess

of homozygotes. The fixation index (ƒ) ranges

from -1 to 1, in which positive values indicate

excess homozygotes and negative values indi-

cate excess heterozygotes (Azevedo, 2007).

Inbreeding is a consequence of the reduction

in the number of reproductive individuals in

the population, which induces self-fertilization

and crossbreeding between related individuals

(Young et al., 1996; Reis et al., 2011). Since S.

mombin is a preferentially allogamous species,

with a low rate of self-fertilization and with

a tendency to self-incompatibility (Carneiro

& Martins, 2012; Oliveira et al., 2012), it is

unlikely that only self-fertilization is respon-

sible for inbreeding, and may be caused by

crossbreeding between related individuals or

by preferential mating.

The way seeds are dispersed may favor the

formation of a spatial genetic structure, with

more closely related individuals (Sobierajski et

al., 2006). It was found during the collections

that there was a large amount of endocarps

under the individuals, as well as young individ-

uals of S. mombin in a nearby radius. The agou-

tis, tapirs, bats and monkeys are considered

dispersants of seeds of S. mombin (Heithaus et

al., 1975; Henry et al., 2000; Smythe, 1970),

however, no studies were conducted on the

distance of seed dispersion performed by these

animals. In this case, if they consume the pulp

of the fruit and leave the endocarp near the

mother tree and the seeds germinate giving rise

to a new individual, a spatial genetic structure

is formed that allows the crossing between

related individuals increasing inbreeding. In

addition, there is the possibility of seeds germi-

nating from fruits that fall close to the mother

plant, without the need for a disperser to inter-

fere with the process.

Another factor that can contribute to this is

pollination or pollinating agents of the species.

Turner et al. (1982) found that when pollinators

perform pollination preferably in neighboring

or nearby plants, high levels of inbreeding are

found, even in plants that have self-incompat-

ibility. Small bees, mainly Apis mellifera and

Scaptotrigona tubiba (Carneiro & Martins,

2012; Oliveira et al., 2012), carry out pollina-

tion of S. mombin. This type of bee calculates

energy expenditures for resource collection

and opts for shorter displacements in search

of energy savings (Bizotto & Santos, 2015).

Thus, pollinators may prefer to forage nearby

plants so as not to increase energy expen-

diture, thus assisting in fertilization among

related individuals.

The mean of the PIC showed that the loci

analyzed were moderately informative, two

being poorly informative (PIC < 0.25), four

moderately informative (PIC ranging between

0.25 and 0.50) and one highly informative

(PIC > 0.50), according to the classification of

Botstein et al. (1980). Although they are not

all highly informative, it was possible, through

these loci, to verify the genetic diversity of S.

mombin and find rare and unique alleles.

The Caceres (CC) and Alta Floresta (AF)

populations presented the highest number

of rare alleles and deserve more attention

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regarding conservation since, according to

Raposo et al. (2007), populations that have a

higher proportion of rare alleles are more sensi-

tive to loss of genetic diversity due to genetic

drift. The AF population also presented a great-

er number of exclusive alleles, thus deserving

greater attention for conservation. In addition

to rare and exclusive alleles, 13 common alleles

of local distribution were identified. These

alleles are also important for the conservation

of the species because, supposedly, they are the

ones that ensure adaptation to specific environ-

ments and, thus, can have great value in cases

of sudden environmental changes or even as a

source of genetic variability for resistance to

attack by pests and diseases (Sebbenn, 2003).

The UPGMA dendrogram demonstrated

that the populations formed two groups, one

with the MR and AF populations and the

other containing the other populations. The

cophenetic correlation showed an association

of 78 % (CCC = 0.78) between the distances

obtained in the dissimilarity matrix and the

cophenetic matrix. According to Rohlf (1970),

CCC values greater than 0.70 reflect good

agreement between matrices. The dendrogram

data were confirmed by the structure grouping,

where AF and MR were again presented in the

same group.

The geographic distance of the populations

is not related to the genetic similarity between

them, which was verified by the Mantel test.

Despite the absence of a relationship between

the genetic structure and the geographical dis-

tances for the species, the presence of exclu-

sive alleles in the populations may indicate

that this differentiation process is underway

and that time and evolutionary mechanisms

have not yet been sufficient to differentiate

populations at geographical levels. In addi-

tion, the species presents alleles with high

frequency, which decrease the differentiation

between populations.

The value of gene flow revealed that popu-

lations are not genetically isolated, since it was

greater than 1. According to Wright (1931),

Nm values greater than 1 are sufficient to avoid

random loss of alleles within the population.

However, this information should be evalu-

ated with care, since due to the high distance

between most populations, current gene flow

via pollen or seed dispersal is unlikely. Thus,

this value may refer to the historical gene flow

between populations over time, according to

the distribution dynamics of the species in the

studied area, which helped to make the allelic

frequencies similar amongst themselves.

The populations of S. mombin in the state

of Mato Grosso present genetic diversity and

are promising sources of genetic resources

that can be selected, collected and propagated.

An example of this is the presence of local

common alleles that indicate adaptation to

specific environments. Furthermore, a study of

genetic divergence between S. mombin geno-

types through morphological characteristics

carried out by Silva et al. (2017a) in the region

where the AF, MR and NA populations are

located has already demonstrated the potential

of some sampled genotypes for use in breed-

ing, due to the presence of variation in traits of

interest. These data reinforce the importance

of maintaining the genetic diversity of the

populations studied.

Genetic diversity is higher at the intrapop-

ulational level, thus, in situ or ex situ conserva-

tion of individuals from each of the studied

populations is proposed, in order to enable the

maintenance of genetic diversity and effective

conservation of S. mombin. It is hoped that

this knowledge regarding the genetic diversity

of S. mombin will assist in the implementa-

tion of conservation strategies, since it reveals

how this diversity is distributed in space and it

signals how the loss of genetic variability has

occurred in the species.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict 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.

1033

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(3): 1023-1036, July-September 2021 (Published Set. 08, 2021)

ACKNOWLEDGMENTS

We are thankful to the funders of this

study. This study was financed in part by the

Coordenação de Aperfeiçoamento de Pessoal

de Nível Superior - Brasil (CAPES) - Finance

Code 001”, Fundação de Amparo a Pesquisa

do Estado de Mato Grosso (FAPEMAT – Proj-

ect: Conservação e uso de espécies vegetais

nativas da região Amazônica com potencial

econômico para região Norte do estado de

Mato Grosso. Process n. 166159/2014) and

Embrapa Agrossilvipastoril.

RESUMEN

Spondias mombin (Anarcadiaceae):

caracterización molecular y conservación

Introducción: El fruto amarillo del jobo o yuplón (Spon-

dias 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 micro-

satélites 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 eva-

luó 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, dendro-

grama 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 intrapo-

blacional (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 poblacio-

nes 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 pro-

misorias para la recolección de germoplasma.

Palabras clave: conservación; recursos genéticos; variabi-

lidad genética; microsatélite; jobo, yuplón.

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