research article using random amplified polymorphic dna to...

Research ArticleUsing Random Amplified Polymorphic DNA toAssess Genetic Diversity and Structure of Natural Calophyllumbrasiliense (Clusiaceae) Populations in Riparian Forests

Evacircnia Galvatildeo Mendonccedila1 Anderson Marcos de Souza2 Faacutebio de Almeida Vieira3

Regiane Abjaud Estopa4 Cristiane Aparecida Fioravante Reis5 and Dulcineacuteia de Carvalho6

1 Departamento de Engenharia Florestal Universidade Federal Rural do Rio de Janeiro 23890-000 Rio de Janeiro RN Brazil2 Departamento de Engenharia Florestal Universidade de Brasılia 70919-900 Brasılia DF Brazil3 Unidade Academica Especializada em Ciencias Agrarias Universidade Federal do Rio Grande do Norte59280-000 Macaıba RN Brazil

4 Klabin Avenida Brasil 26 Bairro Harmonia 84275-000 Telemaco Borba PR Brazil5 Embrapa Florestas 74001-970 Goiania GO Brazil6Departamento de Ciencias Florestais Universidade Federal de Lavras 37200-000 Lavras MG Brazil

Correspondence should be addressed to Fabio de Almeida Vieira vieirafayahoocombr

Received 28 April 2014 Revised 26 July 2014 Accepted 28 July 2014 Published 21 August 2014

Academic Editor Robin Reich

Copyright copy 2014 Evania Galvao Mendonca et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The objective of this study was to assess the genetic variability in two natural populations of Calophyllum brasiliense located alongtwo different rivers in the state of Minas Gerais Brazil using RAPD molecular markers Eighty-two polymorphic fragments wereamplified using 27 primers The values obtained for Shannon index (119868) were 0513 and 0530 for the populations located on themargins of the Rio Grande and Rio das Mortes respectively demonstrating the high genetic diversity in the studied populationsNeirsquos genetic diversity (119867119890) was 0341 for the Rio Grande population and 0357 for the Rio dasMortes populationThese results werenot significantly different between populations and suggest a large proportion of heterozygote individuals within both populationsAMOVA showed that 7042 of the genetic variability is found within populations and 2958 is found among populations(B119878119879= 02958)The analysis of kinship coefficients detected the existence of family structures in both populations Average kinship

coefficients between neighboring individuals were 0053 (119875 lt 0001) in Rio das Mortes and 0040 (119875 lt 0001) in Rio Grande Thiscould be due to restricted pollen and seed dispersal and the history of anthropogenic disturbance in the area These factors arelikely to contribute to the relatedness observed among these genotypes

1 Introduction

Of all the ecosystems that constitute the Brazilian Semidecid-uous Forests riparian forests contribute significantly to theconservation of biodiversity mainly due to the relationshipbetween ecological corridors and ecosystem functioning [1]Because of their interconnectedness riparian forests play acentral role in biogeographical and evolutionary shifts as theyfacilitate seed dispersal [2] In Brazil even though riparianforests are protected by law in the Forest Code (Law 4771 of1965) these environments suffer the consequences of human

activity mainly resulting from fragmentation and degrada-tion The reduction of population sizes and the isolationof populations are direct consequences of anthropogenicactivities resulting in inbreeding and genetic drift [3 4]Understanding the distribution of genetic variability of treepopulations in these areas is fundamental in managementprograms aimed at conservation and the survival of riparianforest species

There are a large number of tree species that occur inriparian forests and as such the choice of the target speciesfor genetic and ecological studies is crucial Among them

Hindawi Publishing CorporationInternational Journal of Forestry ResearchVolume 2014 Article ID 305286 8 pageshttpdxdoiorg1011552014305286

2 International Journal of Forestry Research

Calophyllum brasiliense Camb (Clusiaceae) a Neotropicaltree commonly known as ldquoguanandirdquo stands out as a treespecies that occurs preferentially in soils with high humiditysaturation and that is sensitive to environmental changesand susceptible to local extinction The species is widelydistributed throughout Central and SouthAmerica In Brazilthe wide distribution of C brasiliense ensures its presence inwatersheds of several phytoecological regions

C brasiliense presents both male and hermaphroditicflowers The small ldquopollen flowersrdquo (lt1 cm diameter) aremainly pollinated by halictid bees (Halictidae) Its mediumsized spherical seeds (18ndash23mm diameter) are dispersed bygravity and by phyllostomid bats [5] Bats eat the pulp anddiscard seeds beneath their feeding roosts [6] Seeds canalso be dispersed secondarily by water being carried anddeposited downstream [7] Besides the fact that C brasilienseis a visually attractive species it is also economically impor-tant as it is used for animal feed It has been widely used intraditional medicine and recent studies show that xanthonesfrom C brasiliense can potentially decrease the oxidativedamage produced by some prooxidants [8] The species iscommonly used in reforestation programs for environmentalrestoration because its fruit are attractive to fauna and it is alsoused in tree plantations in areas where the soil is permanentlywaterlogged [9] Due to its various uses and importance Cbrasiliense is currently being exploited unsustainably whichmay eventually alter important genetic parameters in thespeciesrsquo gene pool

Information about the structure and genetic diversity ofpopulations as well as understanding other related factors iscrucial for the development and adoption of more adequatemanagement strategies to ensure its genetic conservationGenetic markers like random amplified polymorphic DNA(RAPD) are valuable in studies assessing genetic variationin plant species when using carefully scored RAPD markers[10 11]This technique is based on the amplification of a DNAfragment which occurs when a pair of nucleotides hybridizesin opposite directions from the target sequence this isachieved through denaturation cycles primer annealing andenzyme extension of Taq polymerase [12] Thus the objectiveof this study is to evaluate inter- and intrapopulation geneticvariability to provide information that can be used in themaintenance and conservation of C brasiliense

2 Materials and Methods

21 Sampled Populations The populations sampled in thisstudy are located along two important rivers in Minas GeraisState the Rio Grande in the municipality of Itumirim(21∘1310158400810158401015840S 44∘4810158401410158401015840W) and Rio das Mortes in BomSucesso (21∘0710158402010158401015840S 44∘4810158402210158401015840W) both at an altitude of850m The riparian fragments included in the study aremade up of semideciduous forest types A total of 120 repro-ductive trees were sampled throughout the study areasYoung leaves were collected and stored at the Tree SpeciesGenetic Conservation Laboratory at the Federal Universityof Lavras (UFLA) at minus80∘C until genomic DNA extractionAll trees had their geographical coordinates recorded with

GPS (Garmin 720) to conduct a spatial analysis of the geneticstructure of the studied populations

22 DNA Extraction and PCR-RAPD Reactions GenomicDNA was extracted from 250mg of young leaves In orderto equalize the concentrations of DNA to 10 ngsdotmLminus1 geno-types were diluted in a buffer according to the formula119881 = (119881119904119862119904) minus (10119881119904)10 where 119881 is the volume of theTE solution (120583L) 119881119904 is the sample volume and 119862119904 is thesample concentration Subsequently samples were submittedto PCR-RAPD reactions The DNA was amplified by theRAPDmethod and reactions were conducted in a GeneAmpPCR system 9700 using 20 ldquoOperon technologiesrdquo primers(California USA) EachRAPD reaction occurred in a volumeof 13 120583L containing 32 ng of genomic DNA 02mM of eachDNTp 4 120583M of individual primer 1 unit of Taq polymerase(Phoneutria) PCR reaction buffer (50mM of Tris pH 8320mM of KCl 2mM of MgCl

2 5 120583gsdot120583Lminus1 of BSA 025 of

Ficol 400 10mM of tartrazine) and ultrapure water A PCR9700 thermocycler was used with the following program45 cycles with a denaturation step of 94∘C for 15 secondsannealing of the primer at 42∘C for 30 seconds extension at72∘C for 30 seconds and a final extension step at 72∘C for5min

Amplification products were separated in 10 (wv)agarose gel in TBE buffer running at 200V for 2 h andstained with ethidium bromide bands were estimated bycomparison with a 250-bp ladder (Gibco BRL) Negativecontrols were consistently used to detect amplification ofbands from contaminated DNA Bands with the samemolec-ular weight and mobility were considered identical The gelswere photographed under ultraviolet light using a BioRadtransilluminator

In order to determine the band frequency in each popu-lation images of the gels were carefully analyzed to constructa phenotype matrix composed of 0 and 1 where 0 corre-sponds to a bandrsquos absence and 1 its presence The bandsthat presented weak coloration and minimal definition werediscarded A locus was considered polymorphic when at leasttwo different bands were detected

23 Data Analysis

231 Identification of the Optimal Number of Markers Inorder to verify that the numbers of polymorphic bands gen-erated by the 20 RAPD primers were sufficient to determinegenetic similarity a bootstrap analysis of 10000 permutationswas conducted using GENES software [13] The squared sumof deviations in relation to the new samples and the stressvalue (119864) indicating the fit between the original matrixand the sampled matrix was calculated The number ofpolymorphic bands is considered precise when the stressvalue is less than 005 [14]

232 Genetic Structure To assess genetic variability thesoftware POPGENE (version 132) was used to calculatethe statistics of genetic variation considering the use ofdominant markers and diploid data [15] Keeping in mind

International Journal of Forestry Research 3

that the software takes into account the dominance of thealleles this program assumes that for each estimate of allelicfrequencies the loci are in Hardy-Weinberg equilibriumTheallelic frequencies were estimated from the square root of thenumber of occurrences of the null genotype (recessive)

Analysis ofmolecular variance AMOVA [15] was carriedout using the software ARLEQUIN version 3512 [16] Thetotal variance is partitioned into covariance components asthey relate to intra- and interindividual andor populationdifferences [17]

233 UPGMA The estimate of genetic similarity betweeneach pair of genotypes was calculated using PC-ORD 414software [17] The values of genetic distance were calculatedbased on Jaccardrsquos coefficient [18] We used dendrograms ofthe unweighted pair-group method with arithmetic average(UPGMA) to represent genetic similarities [19] Group con-sistency was analyzed by calculating the cophenetic corre-lation coefficient among matrices based on the arithmeticcomplement 119889119892

(119894119895)and cophenetic values known as Mantel

test (119885) [20] using NTSYS-pc software version 21 [21] Thistest compares two matrices element by element resulting ina correlation value (119903

119862) that represents the level of relation

among the matrices with the significance of 119903119862tested by

permutations [22]

234 Fine-Scale Spatial Genetic Structure (SGS) Kinshipcoefficients (119865

119894119895) were calculated from RAPD data according

to the dominant marker estimator described by Hardy [23]This coefficient is the probability that two genes drawnrandomly from two individuals are identical by descent thatis the coefficient estimates the genetic coancestry betweenindividuals The estimate of 119865

119894119895with dominant markers

requires the calculation of the inbreeding coefficient 119865119868119878[23] We estimated 119865119868119878 based on allozymic data [24] Theinbreeding coefficient (119865119868119878) wasminus0046 for the Rio dasMortesand 0311 for the Rio Grande Spatial genetic patterns werefurther quantified by the 119878119901 statistic [25] for each populationbased on kinship analyses using the program SPAGeDi v12g [26] The 119878119901 value was estimated as 119878119901 = minus119887log(1 minus119865(1)) where 119887log is the regression slope and 1198651 is the meankinship coefficient between individuals belonging to the firstdistance class A jackknife procedure (across loci) was usedto estimate standard errors for each distance class and 1000randomizations of spatial location were conducted to test foroverall spatial structure under the null hypothesis (119887log = 0)

3 Results

31 Profile of RAPDMarkers Of the total 27 primers selectedinterpretation was possible for only 20 primers resultingin a total 91 and 92 markers for the Rio Grande and Riodas Mortes populations respectively (Table 1) Each primerrevealed from 10 to 70 polymorphic loci producing anaverage of 52 polymorphic loci per primer

32 Evaluating the Optimal Number of Markers In order toevaluate if the number of amplified polymorphic fragments

Table 1 Primers nucleotide sequence total number of loci (NL)and number of polymorphic loci (NPL) for two analyzed Calophyl-lum brasiliense populations

Primer Nucleotide sequence Rio Grande Rio das MortesNL NPL NL NPL

OPB-20 51015840-GGACCCTTAC-31015840 5 5 7 7OPC-10 51015840-TGTCTGGGTG-31015840 3 2 5 5OPE-04 51015840-GTGACATGCC-31015840 3 3 3 3OPE-05 51015840-TCAGGGAGGT-31015840 3 3 5 5OPE-20 51015840-AACGGTGACC-31015840 7 6 6 6OPJ-19 51015840-GGACACCACT-31015840 5 5 3 2OPM-02 51015840-ACAACGCCTC-31015840 4 4 7 7OPN-02 51015840-ACCAGGGGCA-31015840 5 5 3 3OPN-03 51015840-GGGGGATGAG-31015840 5 5 2 1OPN-05 51015840-ACTGAACGCC-31015840 7 7 5 5OPN-16 51015840-AAGCGACCTG-31015840 3 3 4 3OPN-18 51015840-GGTGAGGTCA-31015840 3 3 3 3OPP-18 51015840-GGCTTGGCCT-31015840 4 4 4 4OPP-19 51015840-GGGAAGGACA-31015840 5 5 6 5OPX-01 51015840-CTGGGCACGA-31015840 6 6 6 6OPX-02 51015840-TTCCGCCACC-31015840 6 6 6 6OPX-06 51015840-ACGCCAGAGG-31015840 5 5 5 5OPX-11 51015840-GGAGCCTCAG-31015840 7 7 7 7OPX-12 51015840-TCGCCAGCCA-31015840 4 4 4 4OPX-15 51015840-CAGACAAGCC-31015840 4 4 4 4Total 94 92 95 91

was able to represent the speciesrsquo existent genetic diversity weconducted a bootstrap resampling analysis In this analysiswe used the 92 generated fragments from the Rio Grandepopulation (Figure 1) and 91 fragments generated for theRio das Mortes population (Figure 2) The optimal numberof bands was 81 in both populations With this number offragments the correlationrsquosmagnitude value was very close tothe maximum for the trees sampled in Rio Grande (119903 = 097)and in Rio das Mortes (119903 = 098)

33 Interpopulation Genetic Diversity Neirsquos genetic diversity(119867119890) was 0341 for the Rio Grande population and 0357for the Rio das Mortes population and they were notsignificantly different (Table 2) The estimated values for theShannon index (119868) suggest high genetic diversity within andamongC brasiliense populations with a value of 0513 for RioGrande 0530 for Rio das Mortes and 0588 for the entirepopulation

34 Population Genetic Structure AMOVA showed that7042 of the genetic variability of C brasiliense is foundwithin populations and 2958 among populations with anB119878119879 value of 02958 (Table 3)

35 Genetic Similarity We observed that only individuals 2223 37 and 53 sampled from the Rio Grande population weregroupedwith individuals from the Rio dasMortes population


0

02

04

06

08

1

12

7 17 27 37 47 57 67 77 87 97

Number of fragments

Cor

relat

ion

(r)

SQd = 111

r = 097

E = 0045

Figure 1 Resampling analysis (bootstrap) and the correlation (119903)with number of fragments from the Rio Grande population SQd isthe squared sum of the deviations in relation to the new samples 119864is the stress value

0

02

04

06

08

1

12

7 17 27 37 47 57 67 77 87 97

Number of fragments

Cor

relat

ion

(r)

r = 098

E = 0040

SQd = 078

Figure 2 Resampling analysis (bootstrap) and the correlation (119903)with number of fragments from the Rio dasMortes population SQdis the squared sum of the deviations in relation to the new samples119864 is the stress value

Table 2 Genetic diversity and Shannon index for two Calophyllumbrasiliense populations

Population 119899 Genetic diversity (He) Shannon index (I)Rio Grande 60 0341 0513Rio das Mortes 60 0357 0530Overall 120 0403 0588119899 sample size

(Figure 3) This result underscores the level of divergenceamong trees sampled in both studied populationsThe resultsconfirmed that the polymorphic bands obtained in these twopopulations using 20 RAPD primers were able to assess thegenetic similarity of C brasiliense trees We can thereforeassume that the grouping of the individuals based on thecophenetic correlation is highly reliable (Figure 3)

36 Fine-Scale Spatial Genetic Structure Kinship coefficientanalysis detected the existence of family structures in both

studied populations (Figure 4) The average kinship coeffi-cient between neighboring individuals (represented by 119865

(1))

was 0053 (119875 lt 0001) in the Rio das Mortes populationand 0040 (119875 lt 0001) in the Rio Grande populationindicating that neighboring individuals have a higher levelof genetic relatedness than random pairs of individualsNegative values of 119865119894119895 occurred with increased distancesbetween individuals for both populations Overall slopes(119887log) of two correlograms were significantly different fromthe null hypothesis of no spatial genetic structure (119887log = 0)119887log = minus00223 plusmn 00036 (se) for Rio das Mortes and 119887log =minus00228 plusmn 00040 (se) for Rio Grande 119878119901 values revealedstrong patterns of SGS for the total sample and they weresimilar for both populations (119878119901 = 00236 for Rio das Mortesand 119878119901 = 00238 for Rio Grande)

4 Discussion

41 Genetic Diversity The number of RAPD markers usedto evaluate genetic variability in plants is highly variable andnot dependent on the speciesrsquo level of domestication Pigatoand Lopes [27] when evaluating the genetic variability of fourgenerations of Eucalyptus urophylla identified 86 amplifiedfragments Zimback et al [28] detected 72 polymorphicfragments when analyzing the genetic diversity of Trichiliapallida a Meliaceae climax species Wang et al [29] inves-tigating the genetic structure of Neolitsea sericea found 50polymorphic fragments Torezan et al [30] studying Aspi-dosperma polyneuron using RAPD markers encountered 98polymorphic fragments while Phong et al [31] in studyingthe genetic variability of Dalbergia oliveri a leguminous treeof the Fabaceae family found 24 polymorphic fragmentswhen using 29 RAPD primers Considering the number ofpolymorphic fragments described in the many publishedstudies on natural tree species populations the number offragments obtained in this study of 92 and 91 (Table 2) canbe considered satisfactory enabling an accurate evaluation ofgenetic diversity for this species

The sufficiency of the number of polymorphic bandsfor genetic similarity calculated by resampling showed lowstress values (119864) 0045 and 0040 at Rio Grande and Rio dasMortes respectively (Figures 1 and 2) According to Kruskal[14] stress values lower than 005 are an excellent indicator ofprecisionTherefore the values obtained in our study of 0045for Rio Grande and 0040 for Rio das Mortes respectivelyvalidate the results obtained for number of fragments

The interpopulation genetic divergence indexes obtainedin our analysis demonstrate that the values of He (0341and 0357) were similar to the values obtained in otherstudies of natural tree species populations Torezan et al [30]studying six natural populations ofAspidosperma polyneuronobtained an He of 0299 for adults and seedlings Estopaet al [32] found 119867119890 values ranging from 0299 to 0333 inEremanthus elytropappus while Zimback et al [28] founda variation of 027 to 033 for 119867119890 when studying Trichiliapallida The high genetic diversity within natural populationsofC brasiliense is consistent with other studies of tree specieswhen comparing the values of Shannonrsquos index (119868) wheresimilarmethodologies were used Lacerda et al [33] observed


Table 3 Analysis of molecular variance (AMOVA) for 120 individuals sampled from two populations ofCalophyllum brasiliense using RAPDmolecular markers

Source of variation df SSD Variance component Total 119875 valueAmong populations 1 384866 616959 2958 lt0001Within populations 118 1733533 1469096 7042 lt0001The data show the degrees of freedom (df) sum of squared deviation (SSD) variance component estimates the percentage of total variance contributed toeach component ( total) and the probability (119875 value)

100 75 2550 0

RG1RG2RG44RG38RG3RG16RG24RG27RG28RG4RG21RG19RG25RG26RG34RG35RG42RG36RG39RG14RG15RG40RG41RG10RG11RG12RG32RG33RG59RG54RG56RG8RG18RG7RG29RG60RG20RG43RG47RG50RG51RG31RG48RG52RG49RG5RG30RG13RG17RG6RG9RG55RG58RG57RG45RG46RM1RM15RM6RM33RM34RM7RM13RM9RM35RM36RM11RM32RM14RM19RM45RM43RM17RM25RM20RM21RM37RM55RM56RM57RM44RM48RM18RM24RM23RM8RM16RM29RM42RM60RM52RM12RM26RM41RM51RM50RM53RM2RM38RM3RM4RM5RM54RM58RM59RM10RM22RM27RM47RM46RM49RM30RM31RM39RM40RG23RG53RG22RG37RM28

Genetic identity

rC = 080

Figure 3 Hierarchical cluster analysis of Calophyllum brasiliense genotypes in populations from Rio Grande (RG) and Rio das Mortes (RM)using UPGMA based on data obtained with RAPD RC cophenetic correlation

119868 values ranging from 0301 to 0367 for Plathymenia reticu-lada Torezan et al [30] obtained an 119868 value equal to 0410 foradult individuals of Aspidosperma polyneuron and Zang etal [34] found diversity values of 04382 using RADPmarkersin natural populations of Larix gmelinii Comparatively these

studies suggest that there is strong evidence thatC brasiliensepresents high levels of genetic variability with 119867119890 values of0341 for the Rio Grande population and 0357 for the Riodas Mortes population This can be related to factors suchas insect-pollinated flowers and a predominantly outcrossed


minus006

minus004

minus002

000

002

004

006

535 1128 1925 2738 3415 4837 6658

Distance (m)

Rio das Mortes

Fij

minus006

minus004

minus002

000

002

004

006

Distance (m)206 399 621 884 3374

Rio Grande

Fij

Figure 4 Correlograms of average coancestry coefficients (119865119894119895)

per distance class in two Calophyllum brasiliense populationsConfidence intervals around each 119865

119894119895-value were obtained using a

jackknife procedure across all loci

mating system [24] In the study area however populations ofC brasiliense have been greatly reduced and disturbed thusthe distribution of genetic variability is likely concentrated insmall groups of individuals

42 Population Differentiation and Fine-Scale Spatial GeneticStructure Gillies et al [35] studying the genetic diversity ofSwietenia macrophylla using RAPD markers observed that8743 of the genetic variability is found within popula-tions Lacerda et al [33] observed that 123 of the geneticvariation of Plathymenia reticula is related to differencesamong populations based onAMOVA Trindade andChaves[36] analyzing the genetic structure of natural populationsof Eugenia dysenterica observed that 863 of the geneticvariability is found among populations Zucchi et al [37]analyzing ten populations of Eugenia dysenterica found2703 of variability among populations and 7297 withinpopulations Cardoso et al [38] studying five remaining pop-ulations of Caesalpinia echinata observed that 284 of thegenetic variability is due to geographical differences betweengroups with 296 of the variability occurring among groupsand 42 among individualsThe results obtained in our studyare consistent with that expected for tree species indicatingthat the studied populations ofC brasiliense exhibit consider-able levels of genetic variability within populations (7042)and high levels of divergence (2958) The high geneticdivergence among populations may be related to the spatialaggregation of trees and geographical distance between thestudied populations (approximately 11 km) Although seedsare generally dispersed by bats [39] the genetic differentiation

found in this study may be an indication of restricted seeddispersal

C brasiliense can present an aggregated distributiondepending on the context in which it occurs for examplesmall rock outcroppings Moreover the landscape in thestudy region is highly disturbed and as such the remaining Cbrasiliense populations are restricted to certain areas There-fore the current gene flow in such habitats can be limitedAccording to Wright [40] values of genetic differentiationamong populations ranging between 015 and 005 indicatemoderate differentiation and between 015 and 025 elevateddifferentiation and values greater than 025 can be consideredvery high differentiation Thus the genetic differentiationbetween the two C brasiliense populations can be consideredto be very high as our analysis produced a value of 029

In this study the observation of significantly positivekinship coefficients over a short distance indicates a clearfamily structure that is genetic cluster within the two popu-lations A similar pattern between populations was foundbased on the correlograms The significant SGS found inboth populations suggests restricted seed dispersal of Cbrasiliense a primary factor responsible for the observedSGS of this species [41] The significant genetic clustering atshort distances in both populations likely reflects seedlingrecruitment around the maternal plant [42 43] Accordingto Wright [44] if seed dispersal is localized at the scale ofinvestigation it will result in spatial clustering of geneticallyrelated individuals and the development of significant SGS

The Sp statistic is a robust method to compare fine-scale genetic patterns among plant species independent ofsampling strategies life histories and population densities[25] In comparison to mean values for other species theestimated Sp values in our study (average 119878119901 = 0024) arewithin the range of those reported in predominantly mixedmating (119878119901 = 00372 plusmn 00367) pollen animal-dispersed(119878119901 = 00171 plusmn 00142) and seed gravity-dispersed (119878119901 =00281 plusmn 00166) species [25]

43 Implications for Genetic Conservation Appropriate con-servation and management strategies are necessary in orderto ensure the long-term genetic variability of C brasilienseThe distribution of genetic variation across the landscape is aprime factor to consider in the conservation andmanagementof natural plant populations [45] Our results show theexistence of family structures and a detailed depiction offine-scale genetic variation patterns This spatial informationcan be used to determine future sampling strategies thatensure appropriate distances between sampled genotypesFor example sampling methods for seed banks (ex situconservation) and definitions of the size of an area for insitu conservation can benefit greatly from this informationBased on the correlogram of average 119865119894119895 in local C brasiliensepopulations individuals located within an area of 535m (atRio das Mortes) and 206m (at Rio Grande) are likely to begenetically similar Therefore to optimize sampling designand limit redundant genotypes only individuals locatedat greater distances than 535m or 206m should be col-lected Furthermore samples should be collected from both


populations since our results show that they are significantlydifferent

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank the Fundacao de Amparo aPesquisa do Estado deMinas Gerais (FAPEMIG) for financialsupport The authors also thank Dr Evelyn Nimmo forediting the English of the paper

References

[1] G Viegas C Stenert U H Schulz and L Maltchik ldquoDungbeetle communities as biological indicators of riparian forestwidths in southern Brazilrdquo Ecological Indicators vol 36 pp703ndash710 2014

[2] E Imbert and F Lefevre ldquoDispersal and gene flow of Populusnigra (Salicaceae) along a dynamic river systemrdquo Journal ofEcology vol 91 no 3 pp 447ndash456 2003

[3] P R Aldrich and J L Hamrick ldquoReproductive dominance ofpasture trees in a fragmented tropical forest mosaicrdquo Sciencevol 281 no 5373 pp 103ndash105 1998

[4] D Couvet ldquoDeleterious effects of restricted gene flow infragmented populationsrdquo Conservation Biology vol 16 no 2pp 369ndash376 2002

[5] E Fischer and F A M dos Santos ldquoDemography phenologyand sex of Calophyllum brasiliense (Clusiaceae) trees in theAtlantic forestrdquo Journal of Tropical Ecology vol 17 no 6 pp903ndash909 2001

[6] I Sazima W A Fischer M Sazima and E A Fischer ldquoThefruit bat Artibeus lituratus as a forest and city dwellerrdquo Cienciae Cultura vol 46 pp 164ndash168 1994

[7] M C M Marques and C A Joly ldquoGerminacao e crescimentode Calophyllum brasiliense (Clusiaceae) uma especie tıpica deflorestas inundadasrdquoActa Botanica Brasılica vol 14 pp 113ndash1202000

[8] T Blanco-Ayala R Lugo-Huitron E M Serrano-Lopez etal ldquoAntioxidant properties of xanthones from Calophyllumbrasiliense prevention of oxidative damage induced by FeSO4rdquoBMC Complementary and Alternative Medicine vol 13 p 2622013

[9] R B Torres L A F Matthes R R Rodrigues and H FLeitao-Filho ldquoEspecies florestais nativas para plantio em areasde brejordquo O Agronomico vol 44 pp 13ndash16 1992

[10] M A Gonzalez-Perez P A Sosa and F J Batista ldquoGeneticvariation and conservation of the endangered endemicAnagyrislatifolia Brouss ex Willd (Leguminosae) from the CanaryIslandsrdquo Plant Systematics and Evolution vol 279 no 1ndash4 pp59ndash68 2009

[11] O Ozbek andAKara ldquoGenetic variation in natural populationsof Capparis from Turkey as revealed by RAPD analysisrdquo PlantSystematics and Evolution vol 299 pp 1911ndash1933 2013

[12] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[13] C D Cruz Programa GENESmdashversao Windows UFV VicosaMinas Gerais Brazil 2001

[14] J B Kruskal ldquoMultidimensional scaling by optimizing good-ness of fit to a nonmetric hypothesisrdquo Psychometrika vol 29pp 1ndash27 1964

[15] A Young D Boshier and T Boyle Forest Conservation Genet-ics CSIRO Publishing Melbourne Australia 2000

[16] D S Schineider L Roesslli and L Excoffier Arlequin ver ASoftware for Population Genetics Data Analysis Genetics andBiometry Laboratory University of Geneva Geneva Switzer-land 2000

[17] B S Weir Genetic Data Analysis 2mdashMethods for Discrete Pop-ulation Genetic Data North Carolina State UniversitySinauerAssociates Suderland UK 1996

[18] M R Anderberg Cluster Analysis for Applications AcademicPress New York NY USA 1973

[19] P H A Sneath and R R SokalNumerical TaxonomyThe Prin-ciples and Practice of Numerical Classification W H FreemanSan Francisco Calif USA 1973

[20] B F J Manly Randomization Bootstrap and Monte CarloMethods in Biology ChapmanampHall London UK 2nd edition1997

[21] F J Rohlf ldquoNumerical taxonomy and multivariate analysissystemrdquo New York NY USA version 170 470 pages 1992

[22] N Mantel ldquoThe detection of disease clustering and a general-ized regression approachrdquo Cancer Research vol 27 no 2 pp209ndash220 1967

[23] O J Hardy ldquoEstimation of pairwise relatedness between indi-viduals and characterization of isolation-by-distance processesusing dominant genetic markersrdquoMolecular Ecology vol 12 no6 pp 1577ndash1588 2003

[24] A M Souza Estrutura genetica de populacoes naturais deCalophyllum brasiliense Camb na bacia do alto Rio GrandeUFLA Tese (Doutorado) Lavras Brazil 2006

[25] X Vekemans andO J Hardy ldquoNew insights fromfine-scale spa-tial genetic structure analyses in plant populationsrdquo MolecularEcology vol 13 no 4 pp 921ndash935 2004

[26] O J Hardy and X Vekemans ldquoSPAGeDI a versatile computerprogram to analyse spatial genetic structure at the individualor population levelsrdquoMolecular Ecology Notes vol 2 no 4 pp618ndash620 2002

[27] S M P C Pigato and C R Lopes ldquoAvaliacao da variabilidadegenetica em quatro geracoes de Eucalyptus urophylla ST Blakepor meio do marcador molecular RAPDrdquo Scientia Florestalisvol 60 pp 119ndash113 2001

[28] L Zimback E S Mori P Y Kageyama R F A Veiga andJ R S Mello Junior ldquoEstrutura genetica de populacoes deTrichillia pallida Swartz (Meliaceae) por marcadores RAPDrdquoScientia Florestalis vol 65 pp 114ndash119 2004

[29] Z Wang S An H Liu X Leng J Zheng and Y Liu ldquoGeneticstructure of the endangered plant Neolitsea sericea (Lauraceae)from the Zhoushan archipelago using RAPD markersrdquo Annalsof Botany vol 95 no 2 pp 305ndash313 2005

[30] J M D Torezan R F de Souza P M Ruas C de Fatima RuasE H Camargo and A L L Vanzela ldquoGenetic variability of preand post-fragmentation cohorts of Aspidosperma polyneuronMuell Arg (Apocynaceae)rdquo Brazilian Archives of Biology andTechnology vol 48 no 2 pp 171ndash180 2005

[31] D T Phong V T Hien T T Thanh and D V Tang ldquoCom-parison of RAPD and ISSR markers for assessment of geneticdiversity among endangered rare Dalbergia oliveri (Fabaceae)


genotypes in Vietnamrdquo Genetic and Molecular Research vol 10no 4 pp 2382ndash2393 2011

[32] R A Estopa A M Souza C O M Moura M C G BotrelE G Mendonca and D Carvalho ldquoDiversidade genetica empopulacoes naturais de candeia (Eremanthus erytropapus (DC)MacLeish)rdquo Scientia Florestalis vol 70 pp 97ndash106 2006

[33] D R Lacerda M D P Acedo J P L Filho and M BLovato ldquoGenetic diversity and structure of natural populationsof Plathymenia reticulata (Mimosoideae) a tropical tree fromthe Brazilian Cerradordquo Molecular Ecology vol 10 no 5 pp1143ndash1152 2001

[34] L ZhangHG Zhang andX F Li ldquoAnalysis of genetic diversityin Larix gmelinii (Pinaceae) with RAPD and ISSR markersrdquoGenetics andMolecular Research vol 12 no 1 pp 196ndash207 2013

[35] A C M Gillies C Navarro A J Lowe et al ldquoGeneticdiversity inmesoamerican populations of mahogany (Swieteniamacrophylla) assessed using RAPDsrdquo Heredity vol 83 no 6pp 722ndash732 1999

[36] M D G Trindade and L J Chaves ldquoGenetic structure ofnatural Eugenia dysenterica DC (Myrtaceae) populations innortheastern Goias Brazil accessed by morphological traitsand RAPD markersrdquo Genetics and Molecular Biology vol 28no 3 pp 407ndash413 2005

[37] M I Zucchi J B Pinheiro L J Chaves et al ldquoGenetic structureand gene flow of Eugenia dysenterica natural populationsrdquoPesquisa Agropecuaria Brasileira vol 40 no 10 pp 975ndash9802005

[38] M A Cardoso J Provan W Powell P C G Ferreira andD E de Oliveira ldquoHigh genetic differentiation among rem-nant populations of the endangered Caesalpinia echinata Lam(Leguminosae-Caesalpinioideae)rdquoMolecular Ecology vol 7 no5 pp 601ndash608 1998

[39] M A R Mello N O Leiner P R Guimaraes Jr and PJordano ldquoSize-based fruit selection of Calophyllum brasiliense(Clusiaceae) by bats of the genus Artibeus (Phyllostomidae) ina Restinga area southeastern BrazilrdquoActa Chiropterologica vol7 no 1 pp 179ndash182 2005

[40] S Wright Evolution and the Genetics of Populations Variabilitywithin and among Natural Populations vol 4 University ofChicago Press Chicago Ill USA 1978

[41] J L Hamrick and J D Nason ldquoConsequences of dispersal inplantsrdquo in Population Dynamics in Ecological Space and TimeO E Rhodes Jr R K Chesser and M H Smith Eds pp 203ndash236 University of Chicago Press Chicago Ill USA 1996

[42] B A Loiselle V L Sork J Nason and C Graham ldquoSpatialgenetic structure of a tropical understory shrub Psychotriaofficinalis (Rubiaceae)rdquoTheAmerican Journal of Botany vol 82no 11 pp 1420ndash1425 1995

[43] S Kalisz J D Nason F M Hanzawa and S J Tonsor ldquoSpatialpopulation genetic structure in Trillium grandiflorum the rolesof dispersal mating history and selectionrdquo Evolution vol 55no 8 pp 1560ndash1568 2001

[44] S Wright ldquoIsolation by distancerdquo Genetics vol 28 pp 114ndash1381943

[45] B K Epperson ldquoSpatial patterns of genetic variation withinplant populationsrdquo in Plant Population Genetics Breeding andGenetic Resources AHD BrownMTClegg A L Kahler andB S Weir Eds pp 229ndash253 Sinauer Associates SunderlandMass USA 1990

Submit your manuscripts athttpwwwhindawicom

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Environmental and Public Health

Journal of



EcosystemsJournal of


MeteorologyAdvances in

EcologyInternational Journal of


Marine BiologyJournal of


Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

Advances in


Environmental Chemistry

Atmospheric SciencesInternational Journal of



Waste ManagementJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal of

Geophysics


Geological ResearchJournal of

EarthquakesJournal of


BiodiversityInternational Journal of


ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OceanographyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of Computational Environmental SciencesHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


ClimatologyJournal of


Calophyllum brasiliense Camb (Clusiaceae) a Neotropicaltree commonly known as ldquoguanandirdquo stands out as a treespecies that occurs preferentially in soils with high humiditysaturation and that is sensitive to environmental changesand susceptible to local extinction The species is widelydistributed throughout Central and SouthAmerica In Brazilthe wide distribution of C brasiliense ensures its presence inwatersheds of several phytoecological regions

C brasiliense presents both male and hermaphroditicflowers The small ldquopollen flowersrdquo (lt1 cm diameter) aremainly pollinated by halictid bees (Halictidae) Its mediumsized spherical seeds (18ndash23mm diameter) are dispersed bygravity and by phyllostomid bats [5] Bats eat the pulp anddiscard seeds beneath their feeding roosts [6] Seeds canalso be dispersed secondarily by water being carried anddeposited downstream [7] Besides the fact that C brasilienseis a visually attractive species it is also economically impor-tant as it is used for animal feed It has been widely used intraditional medicine and recent studies show that xanthonesfrom C brasiliense can potentially decrease the oxidativedamage produced by some prooxidants [8] The species iscommonly used in reforestation programs for environmentalrestoration because its fruit are attractive to fauna and it is alsoused in tree plantations in areas where the soil is permanentlywaterlogged [9] Due to its various uses and importance Cbrasiliense is currently being exploited unsustainably whichmay eventually alter important genetic parameters in thespeciesrsquo gene pool

Information about the structure and genetic diversity ofpopulations as well as understanding other related factors iscrucial for the development and adoption of more adequatemanagement strategies to ensure its genetic conservationGenetic markers like random amplified polymorphic DNA(RAPD) are valuable in studies assessing genetic variationin plant species when using carefully scored RAPD markers[10 11]This technique is based on the amplification of a DNAfragment which occurs when a pair of nucleotides hybridizesin opposite directions from the target sequence this isachieved through denaturation cycles primer annealing andenzyme extension of Taq polymerase [12] Thus the objectiveof this study is to evaluate inter- and intrapopulation geneticvariability to provide information that can be used in themaintenance and conservation of C brasiliense

2 Materials and Methods

21 Sampled Populations The populations sampled in thisstudy are located along two important rivers in Minas GeraisState the Rio Grande in the municipality of Itumirim(21∘1310158400810158401015840S 44∘4810158401410158401015840W) and Rio das Mortes in BomSucesso (21∘0710158402010158401015840S 44∘4810158402210158401015840W) both at an altitude of850m The riparian fragments included in the study aremade up of semideciduous forest types A total of 120 repro-ductive trees were sampled throughout the study areasYoung leaves were collected and stored at the Tree SpeciesGenetic Conservation Laboratory at the Federal Universityof Lavras (UFLA) at minus80∘C until genomic DNA extractionAll trees had their geographical coordinates recorded with

GPS (Garmin 720) to conduct a spatial analysis of the geneticstructure of the studied populations

22 DNA Extraction and PCR-RAPD Reactions GenomicDNA was extracted from 250mg of young leaves In orderto equalize the concentrations of DNA to 10 ngsdotmLminus1 geno-types were diluted in a buffer according to the formula119881 = (119881119904119862119904) minus (10119881119904)10 where 119881 is the volume of theTE solution (120583L) 119881119904 is the sample volume and 119862119904 is thesample concentration Subsequently samples were submittedto PCR-RAPD reactions The DNA was amplified by theRAPDmethod and reactions were conducted in a GeneAmpPCR system 9700 using 20 ldquoOperon technologiesrdquo primers(California USA) EachRAPD reaction occurred in a volumeof 13 120583L containing 32 ng of genomic DNA 02mM of eachDNTp 4 120583M of individual primer 1 unit of Taq polymerase(Phoneutria) PCR reaction buffer (50mM of Tris pH 8320mM of KCl 2mM of MgCl

2 5 120583gsdot120583Lminus1 of BSA 025 of

Ficol 400 10mM of tartrazine) and ultrapure water A PCR9700 thermocycler was used with the following program45 cycles with a denaturation step of 94∘C for 15 secondsannealing of the primer at 42∘C for 30 seconds extension at72∘C for 30 seconds and a final extension step at 72∘C for5min

Amplification products were separated in 10 (wv)agarose gel in TBE buffer running at 200V for 2 h andstained with ethidium bromide bands were estimated bycomparison with a 250-bp ladder (Gibco BRL) Negativecontrols were consistently used to detect amplification ofbands from contaminated DNA Bands with the samemolec-ular weight and mobility were considered identical The gelswere photographed under ultraviolet light using a BioRadtransilluminator

In order to determine the band frequency in each popu-lation images of the gels were carefully analyzed to constructa phenotype matrix composed of 0 and 1 where 0 corre-sponds to a bandrsquos absence and 1 its presence The bandsthat presented weak coloration and minimal definition werediscarded A locus was considered polymorphic when at leasttwo different bands were detected

23 Data Analysis

231 Identification of the Optimal Number of Markers Inorder to verify that the numbers of polymorphic bands gen-erated by the 20 RAPD primers were sufficient to determinegenetic similarity a bootstrap analysis of 10000 permutationswas conducted using GENES software [13] The squared sumof deviations in relation to the new samples and the stressvalue (119864) indicating the fit between the original matrixand the sampled matrix was calculated The number ofpolymorphic bands is considered precise when the stressvalue is less than 005 [14]

232 Genetic Structure To assess genetic variability thesoftware POPGENE (version 132) was used to calculatethe statistics of genetic variation considering the use ofdominant markers and diploid data [15] Keeping in mind









permutations [22]






3 Results











0

02

04

06

08

1

12

7 17 27 37 47 57 67 77 87 97

Number of fragments

Cor

relat

ion

(r)

SQd = 111

r = 097

E = 0045


0

02

04

06

08

1

12

7 17 27 37 47 57 67 77 87 97

Number of fragments

Cor

relat

ion

(r)

r = 098

E = 0040

SQd = 078







(1))


4 Discussion







100 75 2550 0


Genetic identity

rC = 080





minus006

minus004

minus002

000

002

004

006

535 1128 1925 2738 3415 4837 6658

Distance (m)

Rio das Mortes

Fij

minus006

minus004

minus002

000

002

004

006

Distance (m)206 399 621 884 3374

Rio Grande

Fij
















Acknowledgments


References




















































Journal of












Volume 2014

Advances in









Geophysics






















permutations [22]






3 Results











0

02

04

06

08

1

12

7 17 27 37 47 57 67 77 87 97

Number of fragments

Cor

relat

ion

(r)

SQd = 111

r = 097

E = 0045


0

02

04

06

08

1

12

7 17 27 37 47 57 67 77 87 97

Number of fragments

Cor

relat

ion

(r)

r = 098

E = 0040

SQd = 078







(1))


4 Discussion







100 75 2550 0


Genetic identity

rC = 080





minus006

minus004

minus002

000

002

004

006

535 1128 1925 2738 3415 4837 6658

Distance (m)

Rio das Mortes

Fij

minus006

minus004

minus002

000

002

004

006

Distance (m)206 399 621 884 3374

Rio Grande

Fij
















Acknowledgments


References




















































Journal of












Volume 2014

Advances in









Geophysics















0

02

04

06

08

1

12

7 17 27 37 47 57 67 77 87 97

Number of fragments

Cor

relat

ion

(r)

SQd = 111

r = 097

E = 0045


0

02

04

06

08

1

12

7 17 27 37 47 57 67 77 87 97

Number of fragments

Cor

relat

ion

(r)

r = 098

E = 0040

SQd = 078







(1))


4 Discussion







100 75 2550 0


Genetic identity

rC = 080





minus006

minus004

minus002

000

002

004

006

535 1128 1925 2738 3415 4837 6658

Distance (m)

Rio das Mortes

Fij

minus006

minus004

minus002

000

002

004

006

Distance (m)206 399 621 884 3374

Rio Grande

Fij
















Acknowledgments


References




















































Journal of












Volume 2014

Advances in









Geophysics

















100 75 2550 0


Genetic identity

rC = 080





minus006

minus004

minus002

000

002

004

006

535 1128 1925 2738 3415 4837 6658

Distance (m)

Rio das Mortes

Fij

minus006

minus004

minus002

000

002

004

006

Distance (m)206 399 621 884 3374

Rio Grande

Fij
















Acknowledgments


References




















































Journal of












Volume 2014

Advances in









Geophysics















minus006

minus004

minus002

000

002

004

006

535 1128 1925 2738 3415 4837 6658

Distance (m)

Rio das Mortes

Fij

minus006

minus004

minus002

000

002

004

006

Distance (m)206 399 621 884 3374

Rio Grande

Fij
















Acknowledgments


References




















































Journal of












Volume 2014

Advances in









Geophysics


















Acknowledgments


References




















































Journal of












Volume 2014

Advances in









Geophysics


































Journal of












Volume 2014

Advances in









Geophysics














research article using random amplified polymorphic dna to...

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