Genetic diversity and structure of Astrocaryum jauari (Mart.) palm in two Amazon river basins

Marcadores microssatélites revelam a diversidade genética e estrutura da palmeira Astrocaryum jauari (Mart.)

ARTICLE

Genetic diversity and structure of Astrocaryum jauari (Mart.) palm in two Amazon river basins

Marcadores microssatélites revelam a diversidade genética e estrutura da palmeira Astrocaryum jauari (Mart.)

Liliane D. Santos Oliveira^I; Santiago L. Ferreyra Ramos^II; Maria T. Gomes LopesI, ^*; Gabriel Dequigiovanni^II; Elizabeth Ann Veasey^II; Jeferson L. Vasconcelos de Macêdo^III; Jacqueline S. Batista^IV; Kyara M. Formiga^IV; Ricardo Lopes^III

^IUniversidade Federal do Amazonas (UFAM), Faculdade deCiências Agrárias, 60.077-000, Manaus, AM, Brazil

^IIUniversidade de São Paulo (USP), Escola Superior deAgricultura “Luiz de Queiroz” (ESALQ), Departamento de Genética, CP 83, 13.400-970, Piracicaba, SP, Brazil

^IIIEmbrapa Amazônia Ocidental, AM 010, CP 319, 69.048-660, Manaus, AM, Brazil

^IVInstituto Nacional de Pesquisas da Amazônia (INPA), Laboratório Temático de Biologia Molecular, Laboratório de FisiologiaComportamental, Coordenação de Biodiversidade, CP 478, 69.060-001, Manaus, AM, Brazil

ABSTRACT

Astrocaryum jauari is a non-domesticated palm that is exploited by poachers. Our objective was to investigate the organization of the geneticdiversity and structure of three A. jauari populations. The study was carried out in the state of Amazonas, between the municipalities of Coari and Manaus. Nine microsatellite loci were used for the genetic analyses. High genetic variation was found, with a mean number of alleles per locus varying from 3.9 to 4.4. The average observed heterozygosity, varying from 0.71 to 0.78, was higher than expected. No spatial genetic structure was detected, since only one cluster was observed. Our results indicate a possible dispersion strategy and suggest that conservation measures of this species should focus mainly on the populations found at the end of the main river (Solimões) where most of the plant material originating from the headwaters of the tributaries of this river is concentrated.

Key words: Amazonia, rivers Solimões and Urucu, Jauari, palm domestication

RESUMO

Astrocaryum jauari é uma palmeira não domesticada e explorada de forma extrativista. O objetivo dotrabalho foi investigar a organização da diversidade genética e estrutura detrês populações de A. jauari . Este estudo foi realizado no estado doAmazonas, entre as cidades de Coari e Manaus. Nove microssatélites foram usadospara as análises genéticas. Foi encontrada alta variabilidade genética com um númeromédio de alelos por loco variando 3, 9 a 4, 4. As heterozigosidades observadasmédias, variando de 0, 71 a 0, 78, foram maiores que os valores deheterozigosidade esperada. Não foi detectada estrutura genética espacial e foiobservado apenas um agrupamento. Os resultados indicam uma possível estratégiade dispersão e sugerem que a conservação desta espécie deve ser realizadaprincipalmente nas populações encontradas no final do rio principal (Solimões), pois contêm a maior parte do material genético proveniente das cabeceiras dosafluentes do presente rio.

Palavras-chave: Amazônia, rios Solimões e Urucu, palmeira Jauari, domesticação de palmácea

INTRODUCTION

Astrocaryum jauari Mart. is anAmazonian palm of the Arecaceae family, adapted to seasonal oscillations inriver levels (Kahn and Millán 1992) that involve a flood phase where watercovers the plains on the margins of the river and a dry phase after the waterrecedes. These plains are therefore referred to as floodplain forests(whitewater-inundated forests are known as várzeas andblackwater-inundated forests as igapós ) (Sioli 1984, Kahn and Millán1992). It is important to mention that the water color is mainly given by theplace of origin and by the sediments that contribute to each of them. Whitewater rivers have their source in the Andes mountain range and containconsiderable amounts of suspended clay, while black waters originate from theforest and are darkened by colloidal humic particles from decomposing organic matter (litter) (Sioli 1984).These seasonal floodings have led to the adaptation of A. jauari andother species to ensure survival when partially or totally submersed duringthose phases (Piedade et al. 2006). The distribution of A. jauari andother plants adapted to this habitat varies according to flooding regimes andseasonality, precipitation patterns around the hydrographic basins and due toevolutionary geographical events, defined by the effect of sediment depositionand erosion on changes in river courses (Albernaz et al. 2012).

A.jauari is found inthe Amazon regions of Colombia, Venezuela, French Guiana, Guiana, Suriname, Ecuador, Peru, and Brazil (Kahn 2008). In Brazil, it occurs in the states ofPará, Acre, Roraima, and Amazonas (Kahn and Millán 1992). This species istraditionally used by the people of different native Amazonian communities as asource of food, fiber and shelter (Zambrana et al. 2007), and until 1998 isalso constituted the basis of industrial heart-of-palm production in CentralAmazonia (Piedade et al. 2003). Furthermore, the fruit of this species is afood source for a variety of fish, especially of the Characidae family. Thisfamily comprises the two most important fish species in terms of localconsumption and exportation: Piaractus brachypomus Cuvier (Pirapitinga)and Colossoma macropomum Cuvier (Tambaqui). This suggests aco-evolutionary symbiosis between A. jauari and these fish species thatfeed on the palm fruits and help disperse the seeds (Piedade et al. 2006).

A.jauari is one ofthe many species of Amazonia about which no information on phenological, agronomic or genetic aspects is available. Knowledge about the diversity andgenetic structure of natural A. jauari populations is an importantinitial step towards the species' conservation and management to initiate adomestication process. It is worth emphasizing that this species is one of the alternativesthat could lead to improvements in the life quality of river populations. Forbeing considered highly important for both mankind and the surrounding faunaand for playing a vital role in the development of local communities, theconservation, management and exploitation of this resource are extremelyimportant.

Similarly to other plant species adapted to the conditions of the floodplains of theUrucu and Solimões rivers, A. jauari occurs in the municipality of Coariin Amazonas where the corporation Petróleo Brasileiro S.A. (Petrobrás) exploresoil and natural gas fields. All crude oil produced by the company istransported by river across these hydrographic basins and the gas through apipeline that consists of two 280 km tubes, beginning in Coari at the RiverUrucu and ending in Manaus, the state capital (Frota et al. 2010). The area ofinfluence of this company includes floodplains (both várzeas and igapós ), dry land and rivers, i.e., unforeseen events could affect the vegetativebiodiversity of the area (Frota et al. 2010).

In addition, the Amazon rainforest has an annual average temperature of 23.5 ºC, annual rainfall of 2464 mm and evapotranspiration of 1657mm (Shukla et al. 1990). However, the Brazilian Amazon has been affected byextreme climatic events in terms of rainfall over the past decade, resulting indrought (and consequent temperature increases) and flooding of rivers. Thiscould be related to the phenomena El Niño and La Niña, whichinfluence the sea surface temperature in the Pacific. Considering thehydrological regime, the Amazon region seems to be affected by events in theAtlantic and Pacific oceans. Changes in the sea surface temperature causechanges in atmospheric dynamics. The variability of atmospheric dynamicsinduces new settings in the patterns of rainfall and flow in the Amazon Basin(Sena et al. 2012). Therefore, given the lack of knowledge regarding thediversity and genetic structure of this species and on how to protect itagainst climatic and man-made events, the objective of this study was toevaluate natural A. jauari populations in the hydrographic basins of theUrucu and Solimões rivers using heterologous microsatellite markers developedfor Astrocaryum aculeatum G. Mey. Considering that the species understudy has not been domesticated yet, one of the goals of this study was togenerate information on the organization of the genetic diversity and structureof three natural populations of A. jauari, which is indispensable forthe management, use and conservation of the genetic resources of this species.

MATERIAL AND METHODS

Study area and plant sampling

The study was conducted in the State of Amazonas between the municipalities ofCoari and Manaus. The species A. jauari is distributed along the marginsof both white and blackwater rivers of the region (Schluter et al. 1993).Therefore, we sought to sample plants from natural populations from differentcommunities along the hydrographic basins of the Urucu and Solimões rivers, considered to be black and whitewater rivers, respectively (Sioli 1984), andsituated within the influence area of the Coari-Manaus gas pipeline. Sampleswere taken from three populations: one from the community Santa Luzia doBuiuçuzinho situated on the River Urucu, and two from the communities Matrinxãand Nossa Senhora das Graças on the Solimões River; Matrinxã lies between theother two communities (Figure 1A).

The large distances between these communities and the location of each one in thehydrographic basins of the Urucu and Solimões rivers were decisive factors inthe selection of the sampling locations. The community Santa Luzia doBuiuçuzinho (lat 04º 11' 59.9” S, long 63º 42' 33” W) belongs to the municipaldistrict of Coari and lies near Lake Coari. This community, founded in 1990, consistsof around 35 families and is situated on a dry land area. The communityMatrinxã (lat 03º 46' 44” S, long 62º 21' 54” W) belongs to the municipality ofCodajás situated on the River Solimões and is close to the point where thisriver meets the River Urucu. It is considered to be a relatively smallcommunity with only seven families. The community Nossa Senhora das Graças (lat03º 20' 37” S, long 60º 35' 34” W) is located on the right margin of the RiverSolimões in a floodplain within the municipality of Manacapuru. It is inhabitedby 65 families whose main source of both subsistence and income is fishing andwho practice family farming. The two latter communities are located onfloodplains (Fraxe et al. 2007). Unrestricted random sampling was carried outin each of the populations selected for the study, identifying 30 plants. Oneleaflet was collected from each plant, stored in silica gel and stored at -20ºC in the Laboratory of Molecular Biology at the National Institute ofAmazonian Research (LTBM-INPA).

DNA extraction and genotyping of microsatellites

DNA was extracted according to the method described by Doyle and Doyle (1990) andquantified by electrophoresis in agarose gel (0.8% w/v) stained with GelRed^TM(Biotium, Hayward, California, USA). Fourteen microsatellitesdeveloped for A. aculeatum (Ramos et al. 2012)were tested. Amplifications by polymerase chain reaction(PCR) were carried out in a total volumeof 10 µL, containing 10 ngof genomic DNA, 1X buffer, 210 µM of each dNTP, 1.5 mM MgCl₂, 0.16 µM forward primer and M13 solution (FAM or NED) (Schuelke 2000), 0.32 µMreverse primer, and 1.05 U Taq DNA polymerase (Promega, São Paulo, Brazil). Theamplification reactions were carried out in two stages: the first consisted ofheating to 68 ºC for 2 min and then 92 ºC for 30 s; followed by 30 denaturationcycles at 92 ºC for 30 s, followed by annealing using specific temperatures foreach primer pair (Table 1) for 35 s and extension at 68 ºC for 35 s. In thesecond stage, 15 denaturation cycles were carried out at 92 ºC for 20 s, followed by annealing at 53 ºC for 30 s and extension at 72 ºC for 30 s, with afinal extension step at 72ºC for 15 min followed by 68 ºC for 30 min (Ramos etal. 2012). The samples were amplified using a VeritiThermal Cycler (Applied Biosystems, Foster City, California, USA). Theamplification products were visualized by electrophoresis in 1.5% agarose gelstained with GelRed (Biotium, Hayward, California, USA). To determine the allelesize, the high-quality PCR products were subjected to capillary electrophoresisin an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, California, USA) with a marker containing fragments of known size, ET-550 ROX size standard(GE Healthcare, Amersham, Buckinghamshire, UK). The genotypes were observed andanalyzed using the GENEMAPPER v4.0 software program (Applied Biosystems, FosterCity, California, USA).

Statistical analysis

The total number of alleles (A ), number of private alleles (A_P ), observed heterozygosity (H_O ), expected heterozygosity (H_E ), inbreeding coefficient (ƒ ), and Hardy-Weinberg Equilibrium (HWE) fromthe exact test and linkage disequilibrium (LD) tests were estimated for eachlocus and in each individual population using GDA software (Lewis and Zaykin2002). The presence of null alleles was verified for each primer usingMICRO-CHECKER software (Oosterhout et al. 2004) with a confidence interval of95% and 10, 000 interactions.

In an attempt to verify the existence of a genetic structure or differentiationbetween the sampled populations, two matrices were calculated using pairwisefixation index (F_ST ) values between the populations andbetween the geographical basins to which they belong. These analyses wereobtained by a significance test with 99, 999 permutationsand adjusting the level of significance with Bonferroni correction (Rice 1989).Wright (1951)'s F statistics were calculated by Weir and Cockerham (1984)algorithms, evaluating the statistical significance based on 20, 000 bootstrap replicates. The Mantel test (Smouseet al. 1986) was performed to analyzethe correlation between the geographic distances between populations and thelinearized genetic distances (F_ST )(Slatkin 1995), and Nei's genetic distances (Nei et al. 1978) among populations. The tests ofsignificance were performed using 100, 000 permutations.Pairwise calculations of F_ST, the linearized F_ST matrix and the Mantel test were carried out with Arlequin v.3.5.1.2 software (Excoffier and Lischer 2010). Nei's genetic distanceswere calculated using GDA software (Lewis andZaykin 2000) and the geographic distance matrix using the earth.dist functionof the Fossil 0.3.7 package from the R project (Vavrek 2011).

In order to determine the genetic structure of the sampled populations, the number ofclusters within the set of accessions evaluated was estimated using theBayesian analysis with the Structure software (Pritchard et al. 2000). Weadopted the Admixture model used for applicability in real populations. Thenumber of clusters (K ) was estimated using ln Pr(X/K) fordifferent values of K (Pritchard et al. 2000). The number of K was defined from 1 to 8 and 20 interactions were carried out for each K with a burn-in of 100, 000 followed by 500, 000 Markov Chain Monte Carlointeractions. The degree of clustering or dispersion of genetic diversitybetween the sampled plants in the three populations wasvisualized using Principal Coordinates Analysis (PCoA), using GenAlEx 6.5 (Peakall andSmouse 2012).

RESULTS

Transfer of heterologousprimers and genetic diversity indices

Of the 14 testedmicrosatellite primers, 12 produced results that were easy to visualize andonly two amplified no products (Aac08 and Aac09). These 12 microsatellite primers were optimized for the speciesunder study and then used for genotyping each sampledaccession. Three of the 12 primers proved monomorphic and weretherefore excluded from the statistical analysis. Among the nine polymorphic loci, the presence of null alleles was only observed in the Matrinxã population atlocus Aac07.

The nine loci confirmed the presence of a large content of geneticinformation with a total of 46 alleles, with 2 to 9 alleles per locus and anaverage of 5.11. Considering the three populations, the number of alleles perlocus varied from 3.9 to 4.4 (Table 1). Loci Aac06 and Aac04 were shown to bethe most polymorphic (varying from 6.0 to 8.0 alleles per locus), while thelocus with lowest polymorphism was Aac03 (2.0 alleles per locus for the threepopulations) (Table 1). Eight alleles were classified as private, four of whichwere observed in Nossa Senhora das Graças, three in Santa Luzia de Buiuçuzinhoand one in the Matrinxã population. The expected heterozygosity (H_E )or genetic diversity for the Nossa Senhora das Graças population varied from0.19 to 0.81, with an average of 0.56. For the other two populations, H_E varied between 0.45 and 0.79, with averages of 0.63 for Santa Luzia do Buiuçuzinho population and 0.58 for Matrinxã population. The values forobserved heterozygosity (H_O ) were higher than H_E for most loci, except for Aac07 and Aac14. The values for H_O varied between 0.20 and 1.0, with averages of 0.78, 0.74, and 0.71 for SantaLuzia do Buiuçuzinho, Nossa Senhora das Graças and Matrinxã, respectively. Onaverage, the inbreeding coefficient or fixation indices were negative and significantly different from zero, indicating that high levels of heterozygotes were observed. The analyses also indicated that HW was not found at loci Aac03, Aac10, Aac12 and Aac13 in any of the three populations, and the same was observed for locus Aac7 in the Matrinxã population. Linkage equilibrium was detected for most of the studied loci, assessed by Fisher's exact test with20, 000 runs, when compared with probabilities equal to or less than 0.00139after standard Bonferroni correction. The linkage equilibrium of the populations in Buiuçuzinho, Nossa Senhora das Graças and Matrinxã was 88.9%, 77.8% and 58.3%, respectively. This result indicates that most loci segregateindependently.

Genetic structure

Pair wise differentiation between all three populations and between the two hydrographicbasins indicated significant but low values (Table 2). From Wright'sF-statistics (Wright 1951), the observed total inbreeding was F_IT =-0.204and intrapopulation inbreeding or inbreeding owing to reproductive systems (F_IS =-0.266)was lower than inbreeding as a result of subdivision (F_ST =0.049), confirming that the diversity is more concentrated within than betweenpopulations. The Mantel test using both the genetic matrices of linearized F_ST and Nei's genetic distances, combined with the population geographic distances demonstrated high butnon-significant correlations, in other words, the test of significance indicatedthat the relationship between the populations is independent of geographicdistances (linearized F_ST, r=0.986, p=0.167; Nei'sgenetic distances, r=0.998, p=0.167), a result that indicates the lowdifferentiation between populations.

When the number of genetic homogeneous populations (K ) between all plants sampledfrom the three populations was estimated by Bayesian analysis using Structuresoftware, only one cluster was observed (K =1, ln Pr(X/K) =-1820.9)among the accessions studied, indicating that there was no population structure(Pritchard et al. 2000). This result indicates that the three populations aregenetically similar. Therefore, data from both Mantel and Structure analysesindicate no structuring between populations, considering the high geneticsimilarity between these three A. jauari populations in this Amazonianregion.

Principal coordinate analysis (PCoA) using Nei's genetic distances, shown in the scatterplot, with both coordinates explaining 45.3% of the total variation observed(Figure 1B), indicated a separation of members of each population, but alsoshowed that the populations are very similar and this result can be related tothe lack of structure found in the Bayesian analysis, suggesting that all ofthese plants belong to a single population.

DISCUSSION

By the SSRs primers designed for A. aculeatum that weretransferred for use in A. jauari high levels of genetic diversity weredetected for this species. The transferability of the primers was possible as aresult of the homology between the genomes of these two species of the samegenus. This homology was also found in the case of SSRs developed forpeach-palm (Bactris gasipaes Kunth) that were transferred for use intucumã palm (Astrocaryum aculeatum ) (Ramos et al. 2011), as well as thespecies cited in the review by Kalia et al. (2011).

High levels of intrapopulationdiversity were found in this study. The genetic diversity parameters related tothe inbreeding indices presented by A. jauari within the populationsstudied suggest that this species is not endogamous, presenting high levels ofheterozigosity. Similar results were obtained in other palm species such as A.mexicanum Liebm. (Eguiarte et al. 1992), Geonoma schottiana Mart.(Silva et al. 2011), Phoenix dactylifera Linn. (Arabnezhad et al. 2012) and Oenocarpusbataua Mart. (Ottewell et al. 2012). This result also suggests that thisspecies has an outcrossing reproductive system, which may be inferred since theapplication of molecular markers in adult plants and their progenies in speciesof the same genus detected this type of reproductive strategy (Eguiarteet al. 1992, Ramos et al. 2011). In this study, privatealleles were observed for all three populations. The identification of privatealleles in populations is useful for application in genetic conservation andcan help identify populations that require a special management (Kalinowski2004). The highest number of private alleles in this study was found in the Nossa Senhora das Graças population, but for better inferences using theseresults in A. jauari more samples must be screened to observe if thereis any variation in the frequency of these alleles.

The three A. jauari populations sampled in the hydrographic basins of theUrucu and Solimões rivers using microsatellite markers presented significantbut low genetic differentiation and a lack of genetic structure amongpopulations, suggesting that the samples represent a single population. Thegenetic structure is highly dependent on the characteristics of the populationand the species, as well as the species' ability to disperse its geneticmaterial, the degree to which a population is isolated, itsself-incompatibility mechanism and allelic diversity (Leducq et al. 2011).

The currents of the River Urucu flow in the direction of the hydrographic basin ofthe River Solimões, which leads to believe that any seeds that are not consumedby the fish of the Characidae family, Piaractus brachypomus (pirapitinga)and Colossoma macropomum (tambaqui), may be transported either by fishor the currents of these rivers and deposited on the banks of both rivers, which would hypothetically establish similar populations in these hydrographicbasins, with low genetic variability between populations through the flow(migration) of plant material. Although the geographic distance between themost distant populations of this study may reach approximately 349 km along thecourse of the hydrographic basins, the low genetic differentiation observedbetween populations supports our hypothesis that plant material is sharedbetween these populations as a result of the dispersal of plant material foundin headwaters of the hydrographic basins. Previous studies on Tabebuiaochracea (Moreira et al. 2009) and Phoenix dactylifera (Hamza et al.2012) provided similar information, reporting low genetic variability betweenpopulations. The Mantel test supported this hypothesis, showing that geneticdistances between populations are independent of geographical distances.

Principal coordinate analysis indicated high genetic similarity between thethree populations in this study. However, small differences between populationswere detected in the cluster analysis, showing the greater similarity betweenthe populations from Santa Luzia do Buiuçuzinho and Matrinxã, which formed a slightly different group from the Nossa Senhora das Graças population. This information suggests that the gene flow through seeds can beinitiated from the most distant populations and is carried along the course ofthe rivers, forming similar populations. Barluenga et al. (2011) suggested thatseed dispersal is important for the determination of the colonization of newsites and migration between neighboring populations, knowing that the majorityof seed species are dispersed by gravity over very short distances. However, the area of seed dispersal may be substantially higher in anemochorous orzoochoric species. When evaluating populations of Silene latifolia Poiret along the hydrographic basin of the river Waal in the Netherlands, theseauthors showed that most of the genetic variation observed (91%) could be foundwithin subpopulations and that clusters formed in PCoA indicated lowdifferentiation between subpopulations. Another study on Tabebuia ochracea (Cham.) Standley carried out along the basin of the river São Francisco showed a single cluster for the populations using Bayesian analysis, and thatpopulations separated by distances of over 10 km and on opposite margins of theriver did not present genetic differences, suggesting that this long-distanceseed dispersal of this species and that this may involve zoochoric dispersal orphysical events associated with the wind and the river (Moreira et al. 2009).

Therefore, taking the results of similar studies into consideration, our investigationshows the high capacity of A. jauari for dispersal of the plant materialby seeds, indicating high genetic similarity between the three populations inthis study for this species. We believe that this dispersal is mainly a resultof the strong currents of the rivers in these hydrographic basins and byzoochoric dispersal. The genetic results indicate that the conservation of thisspecies should be carried out mainly in the populations found at the end of themain river, in our case the river Solimões and the Matrinxã and NossaSenhora das Graças populations, principally the latter, for containing mostof the material originating from the headwaters of this river's tributaries.This information about A. jauari is important for the conservation ofthe species and can be used to determine policies for its management along thehydrographic basins of Amazonia.

ACKNOWLEDGEMENTS

The authors are indebted to the State of Amazonas Research Foundation (FAPEAM)and National Council for Scientific and Technological Development (CNPq) for scholarships.

Received 17 December 2013

Accepted 14 April 2014

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Publication Dates

• Publication in this collection
  25 Nov 2014
• Date of issue
  Oct 2014

History

• Received
  17 Dec 2013
• Accepted
  14 Apr 2014