Diversity and spatial genetic structure of a natural population of Theobroma speciosum (Malvaceae) in the Brazilian Amazon
Juliana de Freitas Encinas Dardengo1*, Ana Aparecida Bandini Rossi2, Bruna Mezzalira da Silva2, Ivone Vieira da Silva3, Carolina Joana da Silva4 & Alexandre Magno Sebbenn5 1. PPG-Bionorte, Perimetral Rogério Silva, 78.580-000, Alta Floresta, Mato Grosso, Brazil; [email protected] 2. Graduate Program in Genetics and Plant Breeding - University of the State of Mato Grosso, Perimetral Rogério Silva, 78.580-000, Alta Floresta, Mato Grosso, Brazil; [email protected], [email protected] 3. Graduate Program in Biodiversity and Amazon Agroecosystems - University of the State of Mato Grosso, Perimetral Rogério Silva, 78.580-000, Alta Floresta, Mato Grosso, Brazil; [email protected] 4. Limnology Research Center, Biodiversity and Ethnobiology of the Pantanal, University of the State of Mato Grosso, Av. Tancredo Neves, 78.200-000, Cáceres, Mato Grosso, Brazil; [email protected] 5. Forestry Institute of São Paulo, Improvement Section and Forest Conservation Genetics, 02.377-000, São Paulo, São Paulo, Brazil; [email protected]
Received 15-X-2015. Corrected 23-III-2016. Accepted 25-IV-2016.
Abstract: 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 natu- ral 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 pollina- tors, 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. Rev. Biol. Trop. 64 (3): 1091-1099. Epub 2016 September 01.
Key words: Amazonia, genetic variability, microsatellite markers, cacauhy.
The national program of biological diver- species. The Conservation Unit (CU) includes sity, that evaluated and identified priority areas headwaters and stretches of important Amazo- for conservation, sustainable use, and benefit- nian rivers, such as Aripuanã, a tributary of the sharing of biodiversity of the Brazilian Ama- Madeira, Juruena, and Teles Pires Rivers, and zon, established 27 ecoregions of the Brazilian tributaries of the Tapajós, and it encompasses Legal Amazon (PNJU, 2013). One ecoregion an area of significant biogeographical inter- is the Mato Grosso dry forests located in the est. The genus Theobroma occurs naturally Northern part of Mato Grosso State. In this in the area. area, the Juruena National Park (Parque Nacio- Several species within the Theobroma nal do Juruena) was established in 2006 with genus have conventional or potential uses, the aim of protecting endemic Amazonian including Theobroma speciosum Willd. ex
Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1091-1099, September 2016 1091 Spreng, commonly known as cacauhy. This Motamayor et al., 2002, Alves et al., 2007, species is important because it represents a Silva et al., 2011). Thus, our aim was to deter- possible source of genetic resistance for other, mine the importance of conservation units, more economically important species, such like the Juruena National Park, in protecting as Theobroma cacao (Silva et al., 2011). The the genetic diversity of tree species of this fruit rind of T. speciosum is mixed with wood area. Using microsatellite markers, we assess ash to produce a handmade soap that is used the genetic diversity and the spatial genetic in the Amazon and it is an excellent deodorant structure (SGS) of a natural population of T. (Di Stasi & Hiruma-Lima, 2002). In relation speciosum in Juruena National Park, as well as to its fatty acids, Gilabert-Escrivá et al. (2002) to provide a census of adults and subadults of T. noted that the composition is very similar to speciosum in the population. We addressed the that found in cocoa butter. Furthermore, Balée following questions: (i) Are there differences (1994) described its use as a source of nutrition in the levels of genetic diversity between adult by Ka’apor indigenous people in the Amazon and subadult populations? (ii) Is genetic diver- region, and its use by the Tacana of Bolivia has sity spatially structured within the population? also been reported (Dewalt et al., 1999). (iii) What is the minimum distance required While some natural populations of Theo- between seed trees to collect seeds for conser- broma species are protected in conservation vation programs? units, such as the Juruena Nation Park, other populations face increasing pressure from for- MATERIALS AND METHODS est fragmentation and exploitation. Studies in other regions of Brazil have shown that forest Study area and sampling: The Juruena fragmentation can have wide ranging impacts National Park (JNP) is a Full Protection Con- on remnant populations, including modifying servation Unit that was created in 2006 and is the size and dynamics of populations, altering administered by the Chico Mendes Institute the composition and dynamics of communities, for Biodiversity (ICMBio). The JNP has an and changing ecosystem processes (Bittencourt area of 195 752 671 ha, 60 % of which is in & Sebbenn, 2009). Therefore, the quantifica- Mato Grosso State, Brazil, and it is distributed tion of genetic diversity, inbreeding, and the across the municipalities of Apiacás (971 935 existence of spatial genetic structure in species ha or 50 % of the park area), Cotriguaçu, and populations helps us to determine measures Nova Bandeirantes. The rest of the conser- needed for genetic conservation by informing vation unit (40 %) is located in Amazonas ways to maximize the genetic diversity in seed State, distributed across the municipalities of collection strategies for ex situ conservation Apuí and Maués (PNJU, 2013). To assess the programs, breeding, and recovery of degraded genetic diversity of T. speciosum, a plot was areas, as well as identify minimum areas for in established in the JNP (North of Mato Grosso situ conservation (Sebbenn et al., 2010). State) on July 8, 2013 (dry season). The plot Microsatellite markers are frequently used has an area of 200 x 160 m (3.2 ha) and is to estimate genetic indexes that provide infor- located within the research plots set up by the mation about evolutionary history, population Program for Research on Biodiversity (PPB- genetic diversity and structure in natural popu- bio). Within this plot, 40 adjacent subplots lations (Sunnucks, 2000; Selkoe & Toonen, of 20 x 40 m (800 m²) were systematically 2006). However, currently, there are very few established (Fig. 1). In the subplots, a total of studies assessing the ecology and genetics 100 individuals of T. speciosum, with diameter of natural species of the Theobroma genus. at breast height (DBH at 1.3 m) greater than 1 Generally, such studies only focus on the cm were sampled, georeferenced (GPS Garmin economically important species, such as T. Etrex®), and measured for DBH. The distribu- cacao and T. grandiflorum (Sereno et al., 2006, tion of trees was not random in the population
1092 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1091-1099, September 2016 Fig. 1. Geographic location of the studied subplots in the Juruena National Park, Brazil.
Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1091-1099, September 2016 1093 and we observed a clear grouping within the calculated using a jackknife procedure across plot (Fig. 1). Leaves at an intermediate stage of all loci. The level of inbreeding among sampled maturation were collected from T. speciosum individuals was estimated using the fixation trees, stored in paper bags, transferred to the index (F) according to the method of Weir and laboratory within two days, and kept at -20º C Cockerham (1984). The significance of the until processing. F values was calculated by permuting alleles among individuals, and sequential Bonferroni DNA extraction and microsatellite anal- correction for multiple comparisons. All analy- ysis: Total DNA extraction was performed fol- ses were run using the FSTAT program, version lowing the CTAB protocol developed by Doyle 2.9.3.2 (Goudet, 1995). and Doyle (1987), with modifications. After The spatial genetic structure (SGS) was extraction, DNA amount and quality were analyzed for three distinct subsamples: (i) all assessed by a comparative analysis of the sam- genotypes (n= 100); (ii) adults (n= 49); and ples on 1 % agarose gel stained with ethidium (iii) subadults (n= 51). The characterization bromide. The samples were diluted in ultrapure of spatial distribution of genotypes inside the water and standardized to 10 ng/mL volumes. population was carried out using the coan-
Twenty-three pairs of microsatellite primers cestry coefficient (θxy) between all pairwise developed for Theobroma cacao (Lanaud et al., individuals within 10 distance classes, based on 1999) were tested for initial amplification using the method proposed by Loiselle et al. (1995): PCR. Five of these primers were selected for the final analysis of all individuals. PCR ampli- fications were carried out according to the fol- lowing protocol: 94º C for 4 min followed by 32 cycles of 94 ºC for 30 seconds, annealing temperature for each locus for 1 min (46 or 51 where, pi and pj are the frequency of k allele ºC), and a final elongation step at 72 ºC for 5 in individuals i and j; pk is the average fre- min (Lanaud et al., 1999). The amplification quency of alleles in the parental population; products were separated by electrophoresis and n is the sample size. To test whether there on 2 % agarose gel in 1X TBE running buffer was significant SGS, the 95 % confidence (89.15 mM Tris base, 88.95 mM boric acid, and interval (95 % CI) was calculated based on the 2.23 mM EDTA) at a constant voltage (80 V) standard error of the mean of the estimates by for 4 hours. The gel was stained with 0.6 ng/ jackknife resampling across loci. Coancestry mL ethidium bromide. The size of the ampli- coefficients and 95 % CI were calculated using fied fragments was estimated by comparison the program SPAGeDi version 1.3a (Hardy with the 100-bp DNA Ladder (InvitrogenTM) & Vekemans, 2002). molecular marker. The amplified fragments were analyzed using the GelQuant Pro program RESULTS to construct a matrix based on fragment size. The genetic diversity of adults (49, DBH> Genetic diversity in adults and sub- 5 cm) and subadults (51, DBH< 5 cm) was adults: The T. speciosum population presented estimated based on the total number of alleles a high level of genetic diversity for the loci (k), average number of alleles per locus (A), analyzed in this study. The number of alleles effective number of alleles (Ae), observed het- per locus ranged from 7 to 13 in adults and erozygosity (Ho), and expected heterozygosity from 7 to 14 in subadults (Table 1). The at Hardy-Weinberg equilibrium (Ho) for each adults presented a similar total number of locus and across all loci. To compare the aver- alleles (52) to subadults (53), with only one age values between adult and subadult trees, exclusive allele found among subadults. The the standard error of these parameters was mean effective number of alleles per locus
1094 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1091-1099, September 2016 TABLE 1 Genetic diversity and inbreeding in microsatellite loci of adult and subadult trees of Theobroma speciosum
Adults (N= 49) Subadults (N= 51) Locus k Ae Ho He F k Ae Ho He F mTcCIR3 13 12.9 0.33 0.91 0.63* 14 14.0 0.60 0.91 0.34* mTcCIR9 7 7.0 0.18 0.79 0.77* 7 6.9 0.23 0.73 0.69* mTcCIR10 12 11.8 0.12 0.91 0.86* 12 12.0 0.15 0.91 0.84* mTcCIR17 10 10.0 0.37 0.90 0.59* 10 10.0 0.42 0.90 0.53* mTcCIR22 10 10.0 0.39 0.90 0.57* 10 10.0 0.46 0.88 0.47* Mean 10.4£ 10.3 0.28 0.88 0.68* 10.6£ 10.6 0.37 0.87 0.57*
SE (95%) ±2.1 ±2.0 ±0.11 ±0.05 ±0.11 ±2.3 ±2.3 ±0.16 ±0.07 ±0.17 Total 52 - - - - 53 - - - -
SE (95%) = calculated by jackknife resampling between loci, *P < 0.05. k = number of alleles; £ = average number of alleles per locus (A); Ae = effective number of alleles per locus; Ho = observed heterozygosity; He = expected heterozygosity; F = fixation index.
(Ae) was also similar to the mean number of genotypes of T. speciosum and 25 genotypes of alleles per locus (A) in adults and subadults. T. subincanum, reported an average of 5 alleles
The observed heterozygosity (Ho) was lower per locus for T. speciosum, and 6.7 alleles per than the expected heterozygosity (He) for both locus for T. subincanum. According to Alves et adults and subadults (Table 1), resulting in sig- al. (2007), most tropical tree species present a nificantly higher than zero fixation index (F), large number of alleles per locus and, conse- indicating inbreeding. quently, a high level of expected heterozygosity. The presence of one private allele in Spatial distribution of genotypes: A the subadult population suggested external strong, significant intrapopulation SGS was gene flow into the study area, meaning the observed for the population (Fig. 2), particu- parents were not located within the sampled larly when either adult or subadult genotypes population. were analyzed separately. In all analyses, the The effective number of alleles per locus coancestry coefficient decreased with increas- (Ae) in adults and subadults underscores the ing distance between individuals, suggesting importance of maintaining conservation units a gene dispersal pattern of isolation by dis- like the JNP in order to preserve species’ tance. This finding indicates that near-neighbor genetic diversity. The loss of rare alleles may individuals are more genetically similar than have a long term impact on the dynamics of expected, based on a random distribution. population genetics, because rare or private alleles represent the potential for adaptation DISCUSSION within the population (Buchert et al., 1997; Rajora et al., 2000).
The number of alleles per locus and the The expected heterozygosity (He) was mean effective number of alleles per locus higher than the observed heterozygosity (Ho). were consistent with the results reported by Alves et al. (2007), studying both natural and Nybom (2004) who reviewed 106 studies of cultivated populations of T. grandiflorum using intraspecific genetic diversity in natural tree microsatellite loci, also found higher values species using microsatellite markers, and found of He (0.42) than Ho (0.35). Similarly, in stud- a mean of 9.9 alleles per locus. On the other ies of T. cacao, Motamayor et al. (2002) and hand, Silva et al. (2015), in their analysis of 25 Sereno et al. (2006) found He (0.540 and 0.566,
Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1091-1099, September 2016 1095 A 0.05 0.04 0.03 0.02 0.01 0 -0.01 23 38 51 63 74 84 95 110 131 208 Coancestry -0.02 -0.03 -0.04 Distance (m) B 0.06 0.04 0.02 0 -0.02 24 41 56 66 76 89 102 117 125 195 Coancestry -0.04 -0.06 Distance (m) C 0.05 0.04 0.03 0.02 0.01 0 -0.01 20 37 49 59 72 82 90 102 119 208 -0.02
Coancestry -0.03 -0.04 -0.05 Distance (m) Fig. 2. Intrapopulation spatial genetic structure in a Theobroma speciosum population. A. All individuals (n= 100); B. subadults (n= 51); C. adults (n= 49). The solid line represents the average θxy value. The dashed lines represent the 95 % (two-tailed) confidence interval of the average θxy distribution, calculated based on the standard error of the mean of the estimates by jackknife resampling across all loci.
respectively) higher than Ho (0.347 and 0.413, Both adults and subadults showed evi- respectively). In contrast, Lemes et al. (2007), dence of inbreeding. T. speciosum is considered studying three Theobroma species using mic- a self-incompatible species (Souza & Ventu- rosatellite loci, obtained different results for rieri, 2010); therefore, inbreeding is likely the result of mating among relatives. This can be He and Ho; in their study, the cultivated T. grandiflorum population had higher H (0.78) explained by the pollen dispersal distances e found for Theobroma species, and particularly than Ho (0.67), whereas natural populations of T. subincanum and T. sylvestris had higher T. speciosum, which is mainly pollinated by Phoridae (Silva & Martins, 2004) and associ- H (0.73 and 0.61, respectively) than H (0.68 o e ated with the presence of SGS. One alternative and 0.43, respectively). In the present study, theory for the high levels of inbreeding is the the evolutionary potential of the population, as presence of Wahlund effect, which results in an demonstrated by Ho, could allow the adaptation increased rate of homozygosity due to the sub- of genotypes to future environmental changes, division of total genetic diversity of a species because of the large number of new genotypic into populations (André et al., 2007). Alves et recombinations that could be generated. Fur- al. (2007) reported a positive fixation index for thermore, the similar values found for A, Ae, natural populations of T. grandiflorum and also Ho and He indicated that subadults and adults noted the possibility of Wahlund effect due to belong to the same gene poll. the possible existence of temporal reproduction
1096 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1091-1099, September 2016 subunits within the sample regions. In our genotypes, Gusson et al. (2005) noted that the study, a strong, significant SGS was observed coancestry estimate (θxy= 0.124) up to 25 m for the population, either when adult and was similar to that expected for half-siblings subadults were analyzed separately or togeth- (0.125), suggesting that the gene flow is limited er. In all analyses, the coancestry coefficient in E. ovata, probably due to short distance pol- decreased with increasing distance between len and seed dispersal. individuals; the results were significant for the Based on the results of the present study, first two distance classes for adults and the total we concluded that seed collection for T. spe- population, and for the first distance class for ciosum should not occur among trees located subadults. This finding indicated that, assum- less than 70 m apart in order to avoid collect- ing random distribution, near-neighbor individ- ing seeds from related seed trees, as this would uals are more genetically similar than expected. reduce the variance effective size (effective An important consequence of the SGS size in the descendant population). However, it detected in many studies of tropical tree species is important to note that even using this strat- (Degen et al., 2004; Hardy et al., 2006) is the egy, the seeds collected may retain some degree common observation of mating among related of biparental inbreeding if mating occurs with- individuals, resulting in an excess of homozy- in the neighborhood of maternal trees, as gotes in relation to the expected Hardy-Wein- observed by Grivet et al. (2009). berg equilibrium. Silva et al. (2011) in their Considering the high demographic density analysis of a natural population of T. cacao in of the species and size of the study area, as Pará State, Brazil, sampled 156 individuals in well as the high average number of alleles per an area of approximately 0.56 ha (278 tree∕ha). locus, we believe that the study population is an They reported a positive and significant SGS excellent resource for in situ genetic conserva- up to a radius of 15 m. The authors associated tion of T. speciosum. The study area is also a the significant spatial correlation to the pres- useful resource for collecting germplasm for ex ence of clones and the aggregated pattern of situ conservation and seed collection, either for individuals within the study area. breeding programs used in the restoration of Ecological and genetic information are degraded areas or forest improvement. essential for understanding a population’s genetic structure. Furthermore, this data informs the development of conservation strat- ACKNOWLEDGMENTS egies and breeding and sustainable manage- The authors wish to thank BIONORTE - ment programs, including defining the size of MT (Projeto Conhecimento, Uso Sustentável reserves, appropriate management of species, e Bioprospecção da Biodiversidade na Amazô- restoration of degraded areas, and seed collec- nia Meridional – Processo: 554330/2010-5) tion for planting native species (André et al., contribution n.º8, PPBio (Projeto Inventário, 2007). Such data are essential for Amazon for- conservação e valoração de alternativas sus- est conservation as they provide key indicators tentáveis do uso da Biodiversidade na Amazô- for the establishment and management of in nia Meridional-Processo: 558319/2009-2) and situ genetic reserves, and inform the develop- CAPES for financial support. We also thank E. ment of gene flow corridors between small R. Nimmo for editing the manuscript. reserves. Based on our estimate of coancestry coefficient, when we consider the entire popu- lation, our analysis detected strong SGS up RESUMEN to a distance of approximately 70 m between Diversidad y estructura genética espacial de una trees. The presence of SGS for T. speciosum is población natural de Theobroma speciosum (Malvaceae) likely the result of short distance seed disper- en la Amazonía brasileña. La cuantificación de la estruc- sal. In their analysis of 108 Eschweilera ovata tura de la diversidad genética e genética intrapoblacional
Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1091-1099, September 2016 1097 espacial (SGS) de especies de árboles son importantes en eastern white pine in Ontario, Canada. Conservation las prácticas de conservación ex situ. En este estudio se Biology, 11, 747-758. busca determinar la importancia de las unidades de conser- vación mediante la cuantificación de la diversidad genética Degen, B., Bandou, E., & Caron, H. (2004). Limited pollen dispersal and biparental inbreeding in Symphonia y la estructura espacial de una población natural de Theo- globulifera in French Guiana. Heredity, 93, 585-591. broma speciosum. Dentro de esta población, a 49 adultos y 51 subadultos se les analizó el genotipo para cinco micro- Dewalt, S. J., Bourdy, G., Michel, L. R. C., & Quenevo, satélites loci. Los resultados mostraron que los adultos y C. (1999). Ethnobotany of the Tacana: quantitative subadultos tienen niveles similares de diversidad genética inventories of two permanent plots of northwestern y endogamia (adultos: A= 10.4, Ae = 10.3, F= 0.68; suba- Bolivia. Economic Botany, 53, 237-260. dultos: A= 10.6, Ae= 10.6, F= 0.57). La diversidad genética detectada fue estructurada espacialmente dentro de la Di Stasi, L. C., & Hiruma-Lima, C. A. (2002). Medicinal plants in the Amazon and Atlantic Forest. UNESP: población y los árboles vecinos hasta una distancia de 70 São Paulo, SP, Brazil. m estaban probablemente relacionados. Esta SGS es proba- blemente el resultado de la dispersión de semillas de corta Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation distancia. El establecimiento de unidades de conservación procedure for small amounts of fresh leaf tissue. permanentes como el Parque Nacional Juruena mostró que Phytochem Bulletin, 19, 11-15. las áreas de conservación son herramientas valiosas en la preservación de la diversidad genética en las poblaciones Gilabert-Escrivá, M. V., Gonçalves, L. A. G., Silva, C. R. naturales. Debido a la alta densidad demográfica de la S., & Figueira, A. (2002). Fatty acid and triacylglyce- especie y el tamaño del área de estudio, así como el ele- rol composition and thermal behavior of fats from seeds of Brazilian Amazonian Theobroma species. vado número promedio de alelos por locus y la presencia Journal of Science Food Agriculture, 82, 1425-1431. de alelos raros, creemos que la población de estudio es un excelente recurso para la conservación genética in situ de Goudet, J. (1995). Fstat. (Version 2.9.3.2.): a computer T. speciosum y también es un recurso útil de germoplasma program to calculate F-statistics. Journal of Heredity, para la recolección de conservación ex situ y la semilla, 86, 485-486. ya sea para programas de mejoramiento utilizados en la restauración de áreas degradadas o mejora de los bosques. Grivet, D., Robledo-Arnuncio, J. J., Smouse, P. E., & Sork, V. L. (2009). Relative contribution of contemporary pollen and seed dispersal to the effective parental Palabras clave: Cacauí, Amazonia, variabilidad genética, size of seedling population of California valley oak microsatélite. (Quercus lobata, Ne´e). Molecular Ecology, doi: 10.1111/j.1365-294X.2009.04326.x.
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