the atlantic rainforest is considered one of the five most important biodiversity hotspots in the world ( myers et al . , 2000 ) and one of the most threatened biomes of the planet . for centuries the atlantic rainforest has been subjected to intense human disturbance that has intensified in the last seven decades , causing the fragmentation of large forest areas and leaving behind , in the majority of its area , only small disconnected forest fragments that are almost entirely isolated and surrounded by extensive anthropogenic matrices , such as pasture , monoculture and areas of urban development ( fundao sos mata atlntica , 2002 ) . hence , the long - term survival of endemic species in this biome will depend on their ability to persist in these environments and our ability to manage and conserve them in degraded landscape ( fahrig , 2002 ) . forest fragmentation exposes populations to ecological and genetic problems caused by inbreeding and loss of variation due to the reduction of effective population size that can lead to genetic drift . the loss of genetic variability that results from such events can cause a decrease in the reproductive ability , disease resistance and genetic plasticity , making it more difficult for natural populations to adapt to environmental change and turning them more susceptible to extinction ( heywood and stuart , 1994 ) . after habitat fragmentation , the majority of species still remains in the fragments for some time , however , the problems caused by the imbalance of the ecosystem favors the dominance of few or a single species . nevertheless , small forest fragments still have significant value for biodiversity , although they are influenced by the size , shape and degree of isolation between them ( turner and corlett , 1996 ) . in this context , studies on animal and plant populations in forest fragments gain increasing importance to address issues like the loss of biodiversity that can cause great harm to future human generations ( wilson and frances , 1997 ) . in order to establish strategies for the conservation of species and ecosystems , further knowledge of the genetic variability in such populations ( leguminosae - mimosoideae ) , is a deciduous , heliophyte , allogamous , monoecious , early secondary tree species that grows on various soil types and is recommended for the recovery of degraded forests , especially in areas of permanent preservation ( durigan and nogueira , 1990 ; vaccaro et al . , 1999 ) . this species is found in the atlantic interior forest of several brazilian states , including minas gerais , so paulo , paran , santa catarina and rio grande do sul , with widest occurrence in the latter three states . it is characterized by a hard wood that is commonly used in construction , shipbuilding , carpentry and joinery , manufacture of coaches , stakes , light and telephone poles and railway sleepers ( lorenzi , 2002 ; da silva et al . , 2012 ) . despite of being a monoecious plant , p. rigida presents self - incompatibility ( ribas la , 1999 , phd thesis , universidade federal de viosa , viosa ) and its seeds are dispersed by wind , water or barochory , and pollen is dispersed by small and medium sized bees ( kageyama , 1992 ) . p. rigida has a lifespan of around 100 years and flowering occurs around 10 years of age ( da silva et al . , 2012 ) . this species also has medicinal properties and is widely used in folk medicine for the treatment of sinusitis , cough ( franco and fontana , 1997 ) , vaginal infections ( korbes , 1995 ) and broncho - pulmonary diseases . gum , resins and tannins can be extracted from its bark and fruit , ( cndido , 1974 ; lorenzi , 2002 ) . these factors make this species very important for restoration programs carried out in the atlantic rainforest . in this study we investigated the genetic diversity within and between p. rigida populations in eight naturally occurring forest fragments of different sizes , using aflp markers in an attempt to determine possible effects of fragmentation on the genetic structure and to provide subsidies for management and conservation of these populations . leaves were collected from 159 adult individuals of p. rigida present in eight forest fragments distributed in eight atlantic rainforest remnants in the southern brazilian states paran and santa catarina . we sampled only individuals that had reached reproductive age , and this was done during their flowering time . the minimum distance among trees was 30 meters , and individuals were sampled throughout all areas of the fragments ( table 1 , figure 1 ) . genomic dna was isolated from approximately 0.5 g of fresh leaves using the ctab method , as described by doyle and doyle ( 1987 ) . the dna concentration was estimated using a fluorometer ( dyna quant 200 , hfer - pharmacia ) , according to manufacturer instructions . briefly , 0,8 to 1.0 g of each dna samples were submitted to restriction digestion by ecori / msei endo - nucle - ases ( 5u each ) and ligation to their respective adapters . after incubation for 16 h at 37 c , the samples were diluted ( 1:10 ) in ultrapure water . polymerase chain reaction ( pcr ) amplifications were carried out using pre - selective primers complementary to the adapters with addition of one 3 nucleotide and diluted 1:10 . for selective amplification , an initial screening was carried out with four individuals from each area using 24 primer combinations . the products of selective amplification were resolved by electrophoresis in polyacrylamide gels ( polyacrylamide 7% acrylamide : bis - acrylamide 29:1 ) for 3 h at 200 v and stained with 20% silver nitrate . a 50-bp molecular ladder ( ludwig biotecnologia , ltda . ) all individuals were scored for the presence or absence of aflp bands ( 1 or 0 ) to construct a binary matrix . only bands with molecular sizes between 100700 bp and only those that could unequivocally scored for presence or absence across all individuals were considered for further analysis . the software dboot v. 1.1 ( coelho , 2001 ) was used to estimate the coefficient of variation ( cv ) for the number of aflp markers , generating a parameter that is capable of determining the reliability of the results obtained with our data . the percentage of polymorphic loci ( pp ) , nei s genetic diversity ( hs ; nei , 1978 ) , the shannon index ( h ) , genetic distance ( nei , 1972 ) , and total heterozygosity ( ht ) were calculated , using popgene v. 1.31 ( yeh et al . , 2000 ) . to test for a correlation between genetic and geographic distances , as well as for a correlation between fragment size and genetic diversity , the pearson s linear correlation and the mantel test were employed using the software bioestat version 5.0 ( ayres et al . , 2007 ) and tfpga version 1.3 ( miller , 1997 ) , respectively . analysis of molecular variance ( amova ) was estimated using arlequin v. 3.11 software ( excoffier et al . , 2005 ) to evaluate the distribu - tion of genetic variation within and among samples , as well as to estimate the fst and pairwise fst indexes . a dendrogram was constructed by means of the upgma method implemented in popgene v.1.3.1 ( yeh et al . , 2000 ) and a bootstrap analysis was done utilizing dboot v.1.1 software ( coelho , 2001 ) . the software structure version 2.3.3 ( hubisz et al . , 2009 the analysis of the number of clusters was performed using the admixture model with a burn - in and run lengths of 10,000 and 100,000 interactions , respectively . the number of clusters was determined following the guidelines of pritchard and wen ( 2004 ) and evano et al . ( 2005 ) , in the online software structure harvester ( earl and vonhodt , 2012 ) . leaves were collected from 159 adult individuals of p. rigida present in eight forest fragments distributed in eight atlantic rainforest remnants in the southern brazilian states paran and santa catarina . we sampled only individuals that had reached reproductive age , and this was done during their flowering time . the minimum distance among trees was 30 meters , and individuals were sampled throughout all areas of the fragments ( table 1 , figure 1 ) . genomic dna was isolated from approximately 0.5 g of fresh leaves using the ctab method , as described by doyle and doyle ( 1987 ) . the dna concentration was estimated using a fluorometer ( dyna quant 200 , hfer - pharmacia ) , according to manufacturer instructions . briefly , 0,8 to 1.0 g of each dna samples were submitted to restriction digestion by ecori / msei endo - nucle - ases ( 5u each ) and ligation to their respective adapters . after incubation for 16 h at 37 c , the samples were diluted ( 1:10 ) in ultrapure water . polymerase chain reaction ( pcr ) amplifications were carried out using pre - selective primers complementary to the adapters with addition of one 3 nucleotide and diluted 1:10 . for selective amplification , an initial screening was carried out with four individuals from each area using 24 primer combinations . the products of selective amplification were resolved by electrophoresis in polyacrylamide gels ( polyacrylamide 7% acrylamide : bis - acrylamide 29:1 ) for 3 h at 200 v and stained with 20% silver nitrate . a 50-bp molecular ladder ( ludwig biotecnologia , ltda . ) all individuals were scored for the presence or absence of aflp bands ( 1 or 0 ) to construct a binary matrix . only bands with molecular sizes between 100700 bp and only those that could unequivocally scored for presence or absence across all individuals were considered for further analysis . the software dboot v. 1.1 ( coelho , 2001 ) was used to estimate the coefficient of variation ( cv ) for the number of aflp markers , generating a parameter that is capable of determining the reliability of the results obtained with our data . the percentage of polymorphic loci ( pp ) , nei s genetic diversity ( hs ; nei , 1978 ) , the shannon index ( h ) , genetic distance ( nei , 1972 ) , and total heterozygosity ( ht ) were calculated , using popgene v. 1.31 ( yeh et al . , 2000 ) . to test for a correlation between genetic and geographic distances , as well as for a correlation between fragment size and genetic diversity , the pearson s linear correlation and the mantel test were employed using the software bioestat version 5.0 ( ayres et al . , 2007 ) and tfpga version 1.3 ( miller , 1997 ) , respectively . analysis of molecular variance ( amova ) was estimated using arlequin v. 3.11 software ( excoffier et al . , 2005 ) to evaluate the distribu - tion of genetic variation within and among samples , as well as to estimate the fst and pairwise fst indexes . a dendrogram was constructed by means of the upgma method implemented in popgene v.1.3.1 ( yeh et al . , 2000 ) and a bootstrap analysis was done utilizing dboot v.1.1 software ( coelho , 2001 ) . the software structure version 2.3.3 ( hubisz et al . , 2009 ) the analysis of the number of clusters was performed using the admixture model with a burn - in and run lengths of 10,000 and 100,000 interactions , respectively . the number of clusters was determined following the guidelines of pritchard and wen ( 2004 ) and evano et al . ( 2005 ) , in the online software structure harvester ( earl and vonhodt , 2012 ) . five selective aflp primers generated 126 polymorphic markers with an average of 25.2 markers per pairwise combinations in 159 individuals that belonged to the eight populations of p. rigida . the ecori - acg / msei - cag and ecori - acg / msei - cat combinations generated the highest ( 28 ) , and the ecori - acg / msei - cta combination the lowest numbers of well defined markers ( 20 ) . the coefficient of variation calculated for the total number of markers was 7.47% , indicating that the number of markers was sufficient to perform the analysess of genetic structure and diversity ( figure 2 ) . so as to verify whether fragmentation had impacted the genetic variability of the populations of p. rigida we calculated the percentage of polymorphic loci ( pp ) , nei s gene diversity ( hs ) and the shannon - wiener index ( h ) , as well as the total heterozygosity ( ht ; table 2 ) . the percentage of polymorphic loci , nei s gene diversity and shannon - wiener index for all populations were pp = 60.4 , hs = 0.217 , and h = 0.322 . when comparing these with the genetic diversities found in other tropical tree species , such as hagenia abyssinica ( hs = 0.30 ; feyissa et al . , 2007 ) , cedrela odorata ( hs = 0.17 ; torre et al . , 2008 ) , aeghilla sellowiana ( hs = 0.10 ; medri et al . , 2010 ) and four other tropical tree species studied by nybom et al . ( 2004 ) ( hs = 0.22 ) , we concluded that most populations of p. rigida still preserve moderate levels of genetic diversity . the populations of curiva and vargem ( table 2 ) showed the lowest percentage of polymorphic loci ( pp = 48.44 and pp = 49.22 ) , nei s gene diversity ( hs = 0.176 and hs = 0.171 ) and the shannon - wiener index ( h = 0.261 and h = 0.257 ) . on the other hand , the populations of ortigueira and irati showed higher values for these genetic parameters ( pp = 82.81 and pp = 79.69 ; hs = 0.301 and hs = 0.287 ; h = 0.444 and h = 0.423 , respectively ) . when comparing the genetic variability of a fragment with their respective sizes a significant correlation between these factors was observed , illustrating the impact of forest cover reduction on the genetic variability of this species ( table 1 ; figure 3b ) . it also needs to be taken into account that in fragmented populations the genetic variability decreases slowly and may be directly correlated to the effective population size . furthermore , according to finkeldey and hattemer ( 2007 ) , the persistence of tree populations in tropical forest over generations is contingent on a minimal population size , as small populations are sooner or later prone to extinction . fragmentation of natural populations can lead to evolutionary constraints due to loss of genetic variability ( frankham , 1996 ; young et al . , these changes are reflected in the processes of genetic drift and gene flow , which determine the degree of genetic diversity of the species ( couvet , 2002 ) . such effects can be observed in the populations of p. rigida , where the populations located in smaller fragments showed an accentuated loss of genetic variability ( tables 1 and 2 ; figure 3b , c and d ) when compared to the populations present in the larger fragments . the analysis of molecular variance ( amova , table 3 ) showed that 72.2% of the genetic variability is distributed within and 22.8% between populations . the fst value ( 0.228 ) indicates a moderate to high genetic variation among populations , which is characteristic of allogamous tree species ( wright , 1969 ; hamrick et al . , 1992 ) such as p. rigida ( monoecious with selfincompatibility ) . such distribution of the genetic variability was also observed by mariot ( mariot a , 2000 , msc dissertation , universidade federal de santa catarina , florianpolis , brazil ) when analyzing four populations of piper cernuum ( fst = 0.29 ) , a species that has a similar reproductive biology as p. rigida . in a further comparison of our results with other studies with natural tropical populations ( paiva , 1998 ) we observed that most of these species preserve high within population genetic variability as observed in p. rigida . in the analysis of pairwise fst ( table 4 ) , the populations of lages and vargem ( 80 km apart ) showed the lowest genetic distance ( fst = 7.43% ) while the populations of vargem and lupionpolis ( 526 km apart ) exhibited the greatest genetic distance ( fst = 32.62% ) . the populations of cornlio procpio and londrina that are distant from each other by only 58 km and the populations of lupionpolis and londrina , distant by 81 km , showed a fst = 17.55% and 25.36% , respectively . interenstingly however , the populations of ortigueira and lages , separated by a geographic distance of 403 km , showed a pairwise fst of only 15.78% . these results show ( table 4 ) that there is no perfect correlation between the geographic and genetic distances ( r = 0.459 , figure 3a ) , an explanation being the different fragmentation intensities that occurred in these areas . historically , the drastic reduction of the atlantic rainforest occurred at different periods in the areas covered by this biome . in the plateau of santa catarina , this event began in the second half of the twentieth century ( vibrans et al . , 2008 ) , whereas in paran , forest fragmentation started in the decade of 1910 and became intensified in the 1950s ( medeiros et al . , 2005 ) . rigida lives around 100 years and flowering occurs around ten years of age . in our samples we collected genetic material from individuals that were already in the reproductive age , with at least one flowering time before sampling . this procedure made it possible to collect only individual that contributed to the real effective population size , even though samples were comprised of individual from many different generations . we constructed a dendrogram using nei s genetic distances ( 1978 ) and th upgma method ( figure 4a ) , showing the separation of two groups that were further confirmed by a bayesian analysis for the k number of clusters ( figure 4b ) . one group was formed by the populations that exhibited the lowest genetic diversity ( curiva , londrina , lupionpolis , and cornlio procpio ) , and a second group that was formed by the populations with the highest genetic diversity ( ortigueira , irati and lages ) , with the exception of the population of vargem that was also present in this second group . the fact that a population such as vargem , that has the lowest values for genetic diversity , clustered very closely with a population such as lages , that presents higher levels of genetic diversity , may be explained by the fact that these populations are historically related and formed a continuous forest until very recently ( around the 1950s ) , and even though the fragment with population of vargen was highly degraded , it still shares much of its genetic diversity with the population of lages . it was also possible to note that the populations with the highest genetic diversity are more closely related to each other ( figure 4a ) , and that the populations with the lowest genetic diversity , that formed a group ( with the exception of vargem ) , are very distant from each other even within their group . such a within group distance might be related to the process of genetic erosion that occurred in these populations , and which is well known to increase genetic distances among natural populations . from this it is possible to conclude that the size of the forest remnants is directly related to the capacity of maintaining higher level of genetic diversity , and that according to holsinger ( 2000 ) , a reduced genetic variation in small populations is likely a symptom of endangerment , and that such populations require immediate management to avoid the extinction of local populations . though reforestation efforts do occur for this species , these are still few and nowhere close to these areas that are considered natural reservoir of genetic variation for this and many other tree species .
parapiptadenia rigida is a tropical early secondary succession tree characteristic of the tropical atlantic rainforest . 
 this species is of great ecological importance in the recovery of degraded areas . in this study we investigated the variability and population genetic structure of eight populations of p. rigida . 
 five aflp primer combinations were used in a sample of 159 individuals representing these eight populations , rendering a total of 126 polymorphic fragments . 
 the averages of percentage of polymorphic loci , gene diversity , and shannon index were 60.45% , 0.217 , and 0.322 , respectively . a significant correlation between the population genetic variability and the population sizes was observed . 
 the genetic variability within populations ( 72.20% ) was higher than between these ( 22.80% ) . 
 no perfect correlation was observed between geographic and genetic distances , which might be explained by differences in deforestation intensities that occurred in these areas . 
 a dendrogram constructed by the upgma method revealed the formation of two clusters , these also confirmed by bayesian analysis for the number of k cluster . 
 these results show that it is necessary to develop urgent management strategies for the conservation of certain populations of p. rigida , while other populations still preserve reasonably high levels of genetic variability .