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Journal of Tropical Ecology (2007) 23:361–367. Copyright © 2007 Cambridge University Press doi:10.1017/S0266467407004099 Printed in the United Kingdom The genetic diversity of Myrciaria floribunda (Myrtaceae) in Atlantic Forest fragments of different sizes Edivani Villaron Franceschinelli∗1 , Giuliana Mara Patrı́cio Vasconcelos†, Elena Charlotte Landau‡, Kátia Yukari Ono§ and Flávio Antonio Maes Santos# ∗ Departamento de Biologia Geral ICB1, Universidade Federal de Goiás, Campus Samambaia, Goiânia, GO 74.001-970, Brazil † BR 010, KM 15, Parada e Hotel Cricabom, Dom Eliseu – PA 68.633-000, Brazil ‡ Departamento de Botânica ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG 3127010, Brazil § Instituto Sócio-Ambiental, São Paulo SP 01238-001, Brazil # Departamento de Botânica, IB, Universidade Estadual de Campinas, SP 13.083-970, Brazil (Accepted 2 March 2007) Abstract: The genetic diversity of Myrciaria floribunda, a common Atlantic Forest tree, was investigated in six populations located in two small fragments (10 and 18 ha), two medium-sized fragments (36 and 44 ha) and one large fragment (3003 ha). Two populations occur in the large fragment. It is expected that smaller fragments should have lower genetic diversity and higher inbreeding. Distances between fragments varied from 0.66 to 10 km. On average 32 young trees smaller than 20 cm basal girth were sampled in each population. Allozyme electrophoresis was carried out, and six loci were scored. The effective number of alleles was lower for populations of the two small fragments (1.46 and 1.51) and higher for populations of the large (1.62 and 1.71) and medium ones (1.69 and 1.84). Small fragments showed lower values of expected and observed heterozygosities than large and medium fragments. Most of the genetic variability occurs within populations, and there was a moderate genetic variation among them (θ̂ p = 0.097). Our findings show a tendency of lower genetic diversity within small and isolated fragments and higher genetic differentiation among them. But, few correlations between genetic diversity indices and fragment features (size and isolation) were significant. Key Words: Brazil, habitat loss, small spatial scale, tropical tree INTRODUCTION Forest fragmentation may cause loss of genetic diversity within populations and disruption of gene flow among populations or subpopulations, leading to depletion of genes that may have adaptive importance (Barrett & Kohn 1991). Habitat fragmentation may decrease reproductive fitness, disease resistance and population adaptation to new environments increasing susceptibility to extinction (Heywood & Stuart 1994, Lienert et al. 2002). Studies showing the effect of habitat loss and isolation on the genetic diversity and gene flow of native species are recent (Bacles et al. 2006, Culley & Grubb 2003, Foré & Guttman 1999, Gonzalez-Astorga & Nunez-Farfan 2001, Hall et al. 1996, Lienert et al. 2002, Prober & Brown 1994, Tomimatsu & Ohara 2003). Some of those studies have 1 Corresponding author. Email: [email protected] been done with native tropical trees (Aldrich & Hamrick 1998, Colevatti et al. 2001, Dayanandan et al. 1999, Dick 2001, Gillies et al. 1999, Hall et al. 1996, Schneider et al. 2003, White et al. 1999). Although many tropical tree populations occur at very low densities and may become isolated after fragmentation, little or no genetic diversity difference has been found between populations of large and small fragments (Lowe et al. 2005). The loss of genetic diversity in tree species is not easily detectable, because they may have originated from mating that occurred before habitat isolation. However, information on the genetic status of native trees that occur in fragmented habitat is important for conducting conservation and reforestation programmes. In Brazil, the Atlantic forest has lost 93% of its primary cover. Its remnants are composed of many small fragments (1–10 ha), several medium-size (more than 30 ha) and a few continuous areas of forest (Tabarelli et al. 1998). However, few studies have been published on the 362 effect of fragmentation on genetic diversity of Brazilian Atlantic forest trees (Auler et al. 2002, Salgueiro et al. 2004, Seoane et al. 2002). Those studies were carried out in a large-scale area, which makes difficult comparative analysis. According to Aguari (2001), the Atlantic Forest was continuous in the study site until 1910, when human activities increased in the area with logging, corn and bean plantations. The main timber exploited was Araucaria angustifolia. The deforestation became more intense after 1950, due to timber exploitation and plantation of exotic Pinus species. At this time, the isolated deforested areas increased in size and number and connected, resulting in the first forest fragments. Those fragments decreased in size until 1985, when timber exploitation and the expansion of agriculture in the area decreased. Nowadays, several fragments are well conserved but others undergo selective logging and cattle grazing. The aim of this study was to assess the variation of population genetic diversity of a tree species within large, medium, and small fragments at a small spatial scale of about 10 km2 . It was conducted in fragments of Atlantic Forest that occur in high altitude (montane forest), located in Southern Minas Gerais State. Myrciaria floribunda (West ex Willdenow) Berg is a common tree species of the Atlantic Forest (Sobral 1993). It may occur in wet forest of Central and South America. It grows slowly and may reach 20 m in height. The species is easy to identify by its whitish bark. Myrciaria floribunda is pollinated by bees and its seeds are dispersed mainly by birds (pers. obs.). It is unknown how fragmentation affects those animals and consequently M. floribunda gene flow among forest fragments. Six populations from five forest fragments were analysed. Two of them are within a quite large fragment (3003 ha), two are in small isolated fragments (10 and 18 ha), and two are in medium-sized fragments (36 and 44 ha). One of the medium fragments is connected to the large fragment. Only leaves of younger plants (basal girth < 20 cm) were collected to increase the chances that the sampled plants were produced after fragmentation. We hypothesize that populations of small isolated fragments have lower genetic diversity than others. Small isolated fragments are expected to have smaller population size, low genetic variation, increased interpopulation genetic divergence due to increased random drift, elevated inbreeding and reduced gene flow. METHODS Study site The study area is located in Serra da Mantiqueira, southern Minas Gerais State, in the municipalities of Camanducaia and Gonçalves, Brazil (Figure 1). The EDIVANI VILLARON FRANCESCHINELLI ET AL. altitude of the studied fragments is between 1500 and 1800 m and the predominant vegetation type is wet montane forest. The matrix surrounding the studied fragments is composed of small farms with pasture, potato and carrot fields. There are no M. floribunda trees in the surrounding matrix. Five fragments were included in the study, one large and four smaller (M1, M2, S1, and S2) (Figure 1, Table 1). Distances between them varied from 0.66 to 10 km. Two distinctive areas, that are 1.18 km apart (L1 and L2), were sampled in the large fragment. L1 area seems to be well preserved, but there is evidence of earlier selective logging. The landowner has a few cattle feeding inside the forest during the dry season, which may affect the understorey plants. L2 area seems better preserved than L1 and it has no indication of recent grazing or logging. M1 fragment covers 44 ha and is not very far from the largest fragment (1.48 km). It has evidence of cattle grazing and selective logging. M2 is 36 ha and is linked to the large studied fragment through a natural corridor of 100 × 30 m (Figure 1). This fragment had been disturbed with selective logging and grazing until 2000, when the new landowner protected it from further disturbance. The two small isolated fragments provide strong evidence of grazing and logging. S1 is 18 ha and it is close to a narrow expansion of the largest fragment (0.66 km). S2 fragment is the most isolated and disturbed. It covers 20 ha, including 10 ha of primary forest, and 10 ha of an old Pinus plantation. Leaves of 28 to 36 plants were collected from the interior of each fragment within an area of about 3 ha. The total sample size was 195 plants. Data on girth at tree base were collected for the sampled plants. Basal girth measure was preferred to girth at breast height, since many trees sampled were smaller than 1.30 m height. Only young plants smaller than 20 cm bg (basal girth) were sampled in order to focus on the generational cohort expected to bear genetic impacts of forest fragmentation. Plant density data were collected along one transect of 50 × 20 m in each studied area. Allozyme analyses The leaf material was transported in liquid nitrogen (−196 ◦ C) from the field to the Universidade Federal de Minas Gerais and stored at −80 ◦ C until enzyme extraction. A small piece of each leaf was ground in liquid nitrogen and the powdered tissue was mixed with extraction buffer number one of Alfenas et al. (1998) and absorbed onto filter paper wicks. Allozyme electrophoresis was carried out on horizontal 13% starch gel. Twenty enzyme systems were screened with three electrophoretic buffers. Five enzyme systems Genetic diversity in Atlantic Forest fragments 363 Figure 1. Map of the sampled fragments in the municipalities of Camanducaia and Gonçalves, Minas Gerais State, Brazil. showed simple banding patterns and could be reliably scored: uridine diphosphoglucose pyrophosphorylase (UGPP, EC 2.7.7.9), isocitrate dehydrogenase (IDH, EC 1.1.1.41), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), triose-phosphate isomerase (TPI, EC 5.3.1.1) and aspartate aminotransferase (GOT EC 2.6.1.1). Uridine diphosphoglucose pyrophosphorylase, IDH, and G6PDH were resolved on a morpholine citrate buffer system at pH 6.5 (Alfenas et al. 1998). Aspartate aminotransferase and TPI were assayed on a lithium borate buffer at pH 8.3 (Soltis et al. 1983). Locus banding patterns were consistent with typical subunit structures. Different loci and alleles for a given system were designated sequentially, with the lowest number corresponding to the most anodally migrating locus or allele. Data analyses Standard measures of allozyme diversity were calculated: the proportion of polymorphic loci (P0.99 ), mean number of alleles per locus (Â) and per polymorphic Table 1. Coordinates and characteristics of the fragments where Myrciaria floribunda were sampled. Isolation degree 1 is the distance of the sampled fragment to the largest fragment (km). Isolation degree 2 is the distance of the sampled fragment to the closest 50-ha fragment (km) and bg is tree basal girth. Fragments L1 L2 M2 M1 S1 S2 Altitude (m) Size (ha) Isolation degree 1 (km) Isolation degree 2 (km) bg of sampled plants (cm) Density (indiv. ha−1 ) 1810 1720 1622 1745 1590 1610 3003 3003 36 44 18 10 0 0 0 1.48 0.66 2.39 0 0 0 0.18 0.66 1.64 15.8 ± 4.13 14.6 ± 4.63 11.2 ± 5.60 13.4 ± 3.72 6.1 ± 2.179 14.9 ± 3.57 49 33 148 150 228 178 364 EDIVANI VILLARON FRANCESCHINELLI ET AL. Table 2. Genetic diversity parameters (mean ± SE) for the studied populations of Myrciaria floribunda. Diversity indices Sample size Expected heterozygosity ( Ĥe ) Observed heterozygosity ( Ĥo ) (all loci) Number of alleles per locus (Â) Number of alleles / polymorphic loci (Âp ) Effective number of alleles (Ae ) Percentage of polymorphic loci ( P̂ ) (0.99) F Wright S1 S2 M1 M2 L1 L2 33.0 ± 2.0 0.28 ± 0.075 0.30 ± 0.082 2.00 ± 0.3 2.33 1.46 83.33 −0.09NS 31.5 ± 0.8 0.30 ± 0.084 0.27 ± 0.07 2.00 ± 0.3 2.33 1.51 83.33 0.09NS 34.5 ± 0.9 0.38 ± 0.078 0.37 ± 0.077 2.00 ± 0.3 2.33 1.69 83.33 0.03NS 30.3 ± 1.1 0.42 ± 0.086 0.34 ± 0.075 2.00 ± 0.3 2.33 1.84 83.33 0.18NS 27.2 ± 0.5 0.35 ± 0.080 0.36 ± 0.092 2.00 ± 0.3 2.33 1.62 83.33 −0.02NS 28.5 ± 0.3 0.38 ± 0.079 0.33 ± 0.068 2.00 ± 0.3 2.33 1.71 83.33 0.13NS locus ( Â p ), effective number of alleles (Ae = 1/ pi2 ), observed heterozygosity ( Ĥo ), the expected heterozygosity ( Ĥe = 1− pi2 ), and Wright’s fixation index F (F = 1− Ĥo / Ĥe ). Test for significant deviations from Hardy– Weinberg expectations was verified through chi-square test within populations. All those parameters were calculated using BIOSYS-2 (original version of Swofford & Selander 1981; modified by Black 1997 – BIOSYS-2: a computer program for the analysis of allelic variation in population genetics and biochemical systematics, release 1.7. Illinois History Survey: 1989, Illinois). Spearman rank order correlations was used to test if parameters of genetic variability ( Ĥe , Ĥo and Ae ) varies according to size and isolation degree of fragments. Wright’s (1943) F-statistics ( fˆ, θ̂ p and F̂ ) were used to measure hierarchical population structure and were calculated by the methods of Weir & Cockerham (1984). Jack-knifing and bootstrapping were used for combining information over alleles and loci, for estimating sample variances and confidence intervals. FSTAT program version 2.9.3.2 (software developed by J. Goudet 2002 – Available from http://www2. unil.ch/popgen/softwares/fstat.htm) was used for these analyses. populations within the medium fragment (0.38 and 0.42, respectively, for M1 and M2). Populations within the large fragment had high heterozygosity values too (0.35 and 0.38, respectively, for L1 and L2). But, t-test showed neither Ĥe nor Ĥo values were significantly different among the studied fragments (α = 0.05). M2 and L2 showed higher fixation indices values (Table 2), but they were not significant (Fisher’s exact test). The correlation analyses revealed that Ĥe , Ĥo and Ae tend to be higher in the largest fragment. Those indices are also higher in fragments closer and connected to the largest fragment of the area. The correlation between the effective number of alleles and fragment size was significant, as well as the inverse correlation between Ĥo and distance from the largest fragment (Table 3). The inverse correlation between Ĥe and distance from the largest fragment was high (r = 0.80) but not significant (P = 0.055). The correlations between Ĥe and area, Ĥo and area, and Ĥo and distance from the closest neighbouring fragment ≥ 50 ha were also high but not significant. F-statistics indicate moderate genetic divergence among the six populations analysed (Table 4). Yet, value of θ̂ p for the smallest and isolated fragments is higher than for populations of the large and connected fragment (Table 5). RESULTS Six loci and twelve alleles were analysed. Significant deviations from Hardy–Weinberg expectations were detected only for the L2 population in the GOT locus. No other loci showed significant deviation from the genotypic proportion expected by the Hardy–Weinberg equilibrium. The percentage of polymorphic loci and average number of alleles per locus were similar for all populations (Table 2). The effective number of alleles (Ae ) was lower for populations of the smallest fragments (1.46 and 1.51) and higher for populations in the largest (1.62 and 1.71). The medium fragment M1 had also high Ae value (1.69). But, the medium fragment M2 (connected) had the highest Ae value (1.84). The average effective number of alleles for the six populations was 1.64. The expected heterozygosity was also low for the two smallest fragments (0.28 and 0.30) and highest for Table 3. Correlation analyses (Spearman Rank Order Correlations) between genetic indices of diversity and characteristics of the studied fragments. Ĥe is expected heterozygosity, Ĥo is observed heterozygosity, Ae is effective number of alleles, area is the size of the fragment in hectares, DLF is distance from the larger fragment, DF 50 ha is distance from the closest neighbouring fragment ≥50 ha. Parameters r P Ĥe and area Ĥe and DLF Ĥe and DF 50 ha 0.75 −0.801 −0.678 0.086 0.055 0.139 Ĥo and area Ĥo and DLF Ĥo and DF 50 ha 0.783 −0.880 −0.759 0.066 0.021 0.080 Ae and area Ae and DLF Ae and DF 50 ha 0.928 −0.577 −0.698 0.008 0.231 0.123 Genetic diversity in Atlantic Forest fragments 365 Table 4. Estimates of Wright’s F statistics (f̂, F̂ and θ p ) described for each polymorphic locus and for overall populations of Myrciaria floribunda. f̂ = mean fixation index of individuals relative to their population; θ p = population coancestrality coefficient; F̂ = mean overall inbreeding coefficient of an individual. Loci fˆ F̂ θ̂ p UGPP-1 IDH-1 G6PDH-1 GOT-2 GOT-3 Mean SE P 0.052 0.186 0.108 −0.017 −0.017 0.189 0.296 0.194 0.050 0.024 0.143 0.139 0.101 0.066 0.040 0.062 0.040 ns 0.153 0.053 ns 0.097 0.021 ns Table 5. Estimates (mean ± SE) of Wright’s F statistics (f̂, F̂ and θ p ) described for the three isolated and the connected fragment populations. F statistics fˆ F̂ θ̂ p Populations isolated Populations connected 0.020 ± 0.058ns 0.137 ± 0.096ns 0.117 ± 0.050ns 0.111 ± 0.031ns 0.158 ± 0.047ns 0.053 ± 0.023ns Plant density of M. floribunda varied from 49 to 228 plants ha−1 . It was negatively related to the fragment size (r2 = 0.84, y = −0.045x + 178, P = 0.0102). Among the sampled plants, fragment S1 showed the smallest average basal girth (6.11 ± 2.17 cm). Fragments L1, S2 and L2 showed the highest average basal girth (Table 1). DISCUSSION Myrciaria floribunda showed high number of alleles per locus (2.00), expected heterozygosity (0.28–0.42) and percentage of polymorphic loci (83.33) compared to the values found for others tropical species. According to Hamrick & Godt (1989), the mean number of alleles per locus and values of expected heterozygosity found for tropical species was 1.45 and 0.109, respectively. The genetic diversity found for M. floribunda is similar to other long-lived, non-endemic tropical Brazilian species (Auler et al. 2002, Conte et al. 2003, Margis et al. 2001, Salgueiro et al. 2004, Seoane et al. 2002). Genetic diversity indices did not differ significantly among the studied fragments. But Ĥe , Ĥo and Ae tended to be lower in smaller and isolated fragments (Table 2). The correlation rates between those indices and fragment size and isolation were high, but only significant in some cases (Table 3). The lack of significant correlations showed above may be explained by the recent forest fragmentation events in the study area. Probably, not enough time has passed for the genetic drift to decrease the genetic diversity of M. floribunda in the small fragments. Some genetic diversity of fragments S1 and S2 may have been lost at the time of fragmentation due mainly to genetic bottlenecks. A single data source on M. floribunda growth rate available in the literature (Laurance et al. 2004) showed a median diameter growth rate of 0.39 mm y−1 . Considering this growth rate and that studied fragments are about 50 y old, a M. floribunda sapling would have girth at breast height of 9.26 cm and basal girth of 11 cm after 50 y. This means that part of our sample may be older than the forest fragmentation in the area. Nevertheless, all plants sampled within S1 were very small trees, smaller than 11 cm bg. Fragments M1 and M2 also had most of their sampled trees smaller than 11 cm bg and these fragments showed high genetic diversity. Inbreeding could have already been detected in these fragments. However, fixation index did not differ among them, besides it was negative and not significant in S1. Other neotropical studies (Apsit et al. 2001, Dick 2001, White et al. 2002) suggest that pollen flow increases under fragmentation, but it depends on the pollinator behaviour after fragmentation. Gene flow among fragments may be preventing such inbreeding effects. According to Mills & Allendorf (1996) and Wang (2004), the migration of 1–10 individuals per generation among fragments may avoid inbreeding. Myrciaria floribunda is pollinated by bees and has its seeds dispersed mainly by birds (pers. obs.). It is not known how fragmentation has affected those animals in the studied area. But, apparently some birds that disperse M. floribunda seeds seem to be able to leave the forest and fly in open areas. Bacles et al. (2006) showed that seed dispersal is up to six times more effective than pollen dispersal at maintaining genetic connectivity among remnants of Fraxinus excelsior, a wind-pollinated and -dispersed temperate tree. We believe gene flow after fragmentation may have increased in the studied sites only in the last 20 y, when Atlantic Forest remnants in the area started being better protected, and as a result, some fragments have increased in size (Aguari 2001). Myrciaria floribunda plant density was negatively related to the fragment size. This may influence the relative low genetic diversity in the large fragment, particularly compared to the medium-sized fragments. The highest values of expected heterozygosity and effective number of alleles were found in the fragment M2. This fragment showed high plant density and is connected to the largest studied fragment, which may increase the access of pollinators and seed dispersers among them. No conclusive explanation was found to explain the lowest plant density in populations of the largest fragment, since M. floribunda seems to be a late-successional species and not expected to do better in disturbed habitat. Aldrich & Hamrick (1998) have found higher abundance and density of Symphonia globulifera seedlings in remnant fragments than in continuous forest, although this is a late-successional species as well. 366 F-statistics showed results similar to those of other tropical tree species (Auler et al. 2002, Eguiarte et al. 1992, Margis et al. 2001, Salgueiro et al. 2004, Seoane et al. 2002). Most of the genetic variability is within populations, but some genetic variation among them (θ̂ p = 0.097) was found. According to Hartl (1988), θ̂ p values of 0.05–0.15 indicate moderate genetic differentiation. However, Wang (2004) presumes that populations are expected to retain genetic connectivity when θ̂ p is smaller than 0.20. In this case, θ̂ p values found for M. floribunda populations may still partly reflect pre-fragmentation landscape conditions. Nevertheless, the genetic variation among populations within the large fragment was lower (θ̂ p = 0.053) than among populations of the small fragments (θ̂ p = 0.117), which indicates that fragmentation may already be increasing differentiation among populations in the studied area. A considerable rate of inbreeding was found at the species level ( F̂ = 0.153). Even populations within the large fragment ( F̂ = 0.158) showed substantial inbreeding. This may be due to habitat loss in the small fragments and genetic differentiation among them, and to the low plant density in the largest fragment. The lack of significant differences in genetic diversity among fragments may also be due to low allele diversity of allozyme markers, which may not provide enough statistical power to test the proposed hypotheses. A new study that includes M. floribunda and other species, higher number of fragments, and DNA markers has been developed in the same area in order to check the present data. ACKNOWLEDGEMENTS This work was supported by a grant from PROBIO/MMA (Projeto de Conservação e Utilização Sustentável da Diversidade Biológica Brasileira/ Ministério do Meio Ambiente) to the first author. We are indebted to Roselaini M. do Carmo, Antonio Pereira da Silva, and Virginia Tenório for their assistance in the lab and in the field. We are grateful to Altair Rezende de Souza, Sebastião Loreano, Zé Maria Pinheiro de Souza, Carlos Alberto Pompeu, Antonio Bueno de Souza e José Leite de Mello for allowing us to work in their properties. Thanks are due to Alexandre Siqueira Guedes Coelho for his help in statistical analyses. The comments of Claudia Jacobi and anonymous revisers greatly improved this final version. LITERATURE CITED AGUARI, 2001. Ações sociais para a preservação de fragmentos florestais na bacia do Rio Camanducaia. Camanducaia: PROBIO/MMA. 40 pp. EDIVANI VILLARON FRANCESCHINELLI ET AL. ALDRICH, P. R. & HAMRICK, J. L. 1998. 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