The Journal of Heredity 2002:93(5)
© 2002 The American Genetic Association 93:365-369
Genetic Diversity in Natural Populations of Piper cernuum
From Nú cleo de Pesquisas em Florestas Tropicais, Universidade Federal de Santa Catarina, CxP 476, Florianópolis 88040-900, Santa Catarina, Brazil (Mariot and dos Reis) and Departamento de Farmacologia, Instituto de Biociências, UNESP, CxP 510, Botucatu 18618-000, São Paulo, Brazil (Di Stasi).
Address correspondence to Maurício Sedrez dos Reis, Departamento de Fitotecnia, Universidade Federal de Santa Catarina, CxP 476, Florianópolis 88040-900, Santa Catarina, Brazil, or e-mail: msreis{at}cca.ufsc.br.
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Abstract |
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Piper cernuum is a native plant of the Brazilian Atlantic rain forest. This work studies the distribution of allozyme diversity in P. cernuum natural populations in order to establish a strategy for sustainable management and conservation. Leaf samples were collected in two Brazilian states. High divergences among populations (FSR = 0.380) and low divergences among regions (FRT = -0.069) and among gaps of the same population (FGT = 0.062) were found. No association between the geographical variation and the genetic distance was detected. An excess of heterozygotes was detected in the populations (FIS = -0.170), suggesting selection in favor of heterozygotes. The results, and the fact that the species depends on constant gap formation for maintenance of its dynamism, suggest that the founder effect is largely responsible for the structuring of populations. For sustainable management, the maintenance of plants/reproductive branches in the gaps is of major importance. The genotypes produced in these gaps are responsible for the establishment of new gaps and are the foundation for new populations, maintaining the dynamics of allele movement.
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Introduction |
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Pariparoba (Piper cernuum Vell.; Piperaceae), a native plant of the Atlantic rain forest (ARF), has been commonly used in folk medicine (Stasi et al. 2002). However, its leaves are extracted from the forest without sustainability criteria. It is a relatively common understory shrub in secondary and primary forests, and the maintenance of this species depends on constant small gap formation for seed germination and establishment, although flowering and seed production continue long after forest regeneration within the gap (Mariot 2000). Seed dispersal in P. cernuum is performed by bats (Carollia perspicillata; Mariot 2000), which can transport its fruits over long distances (Fleming et al. 1977).
Sustainable management can be an option for continuous exploitation if it is compatible with conservation of this ecosystem. Sustainable management has, as a basic premise, control over the process of exploitation, seeking to meet social and economic needs but also seeking, more importantly, the maintenance of the forest resource for the continuity of its use (Reis 1996). Additionally, the guarantee of continuity of any exploitative process is related to maintenance of the genetic structure of the natural population of the species under management. Oyama (1993) and Reis et al. (2000) call attention to the fact that effective actions on conservation and management of tropical forests are still limited, and two basic aspects of population biology should be studied in order to provide a basis for these actions: demography and genetics.
The study of genetic structure and diversity permits knowledge of the organization and distribution of the genetic variability among and within natural populations (Bawa 1992; Hamrick et al. 1992; Reis et al. 2000). This understanding is indispensable in the choice of strategies for conservation and exploitation of natural populations in their natural habitats when the objective is the maintenance of diversity and guarantee of sustainability (Nason and Hamrick 1997; Oyama 1993; Reis et al. 1998, 2000; Sebbenn et al. 2000).
To manage and conserve natural populations of P. cernuum in the ARF more effectively, genetic information about the species needs to be obtained. Here we present allozyme variation in four natural populations of P. cernuum in the ARF.
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Materials and Methods |
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Field Collections
Studies were carried out on four natural populations of P. cernuum in the counties of Sete Barras (Parque Estadual Intervales [PEI]; 24°13' S, 48°05' W), Eldorado (Fazenda Colônia Nova Triestre [CNT]; 24°23' S, 48°15' W), Ibirama (Floresta Nacional de Ibirama/IBAMA [FNI]; 27°02' S, 49°27' W), and São Pedro de Alcântara (Estação Experimental de São Pedro de Alcântara [SPA]; 27°30' S, 48°30' W), all located in the Brazilian ARF. The first two areas (PEI and CNT) are adjacent, without forest fragmentation, located in the Vale do Rio Ribeira do Iguape region, State of São Paulo (SP), and are situated 350 km north of the other two areas (FNI and SPA). FNI is located in the Médio Vale do Rio Itajaí-açu region, State of Santa Catarina (SC), 60 km north of SPA (SC), without connection (caused by forest fragmentation). Mature leaves were collected from 29 reproductive shrubs in PEI, 24 in CNT, 33 in FNI, and 22 in SPA. Samples were put into plastic bags in ice containers and taken to the laboratory, where they were stored at 5°C until needed.
Laboratory Procedures
The populations were analyzed by horizontal starch-gel electrophoresis (13%). The enzymes 6-phosphogluconate dehydrogenase (locus Pgdh-1; EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6pdh-1; EC 1.1.1.49), acid phosphatase (Acp-1; EC 3.1.3.2), shikimate dehydrogenase (Skdh-1; EC 1.1.1.25), and esterase (Est-1; EC 3.1.1.1) were assayed using a tris-citrate buffer system (pH 7.5), and the enzymes peroxidase (Po-1 and Po-2; EC 1.11.1.7), malate dehydrogenase (Mdh-1; EC 1.1.1.37), and malic enzyme (Me-1; EC 1.1.1.40) were assayed using a citrate-morpholine buffer system (pH 6.1) (Alfenas et al. 1991; Cheliak and Pittel 1984).
Statistical Analyses
The data were analyzed using BIOSYS-1 (Swofford and Selander 1989) to obtain the mean number of alleles per locus (A), percentage polymorphic loci (P), observed and expected heterozygosities based on Hardy–Weinberg expectation (Ho and He), and cluster analysis based on genetic distance (Nei 1978) using the unbiased identity method (UPGMA). Genetic structure within and among populations was estimated using F-statistics (Wright 1951), which measure departures from expected levels of heterozygosity. An unbalanced nested model was used, as described by Weir (1990). The F-statistics were used to partition total inbreeding (Wright's FIT) into components resulting from inbreeding within populations (FIS) and subdivision between populations (FSR) and between regions (FRT).
P. cernuum is a typical forest gap species (Mariot 2000). Seeking to detect genetic differences among individuals originating from different gaps in the same population, genetic structure within and among gaps was also estimated using F-statistics (Wright 1951). It used individuals from two gaps (8 and 13 individuals) in CNT population. An unbalanced nested model was also used, as described by Weir (1990). The F-statistics were used to partition total inbreeding (FIT) into components resulting from inbreeding within gaps (FIG) and subdivision between gaps (FGT).
The analyses of the variance (ANOVAs) in the two previously described situations were accomplished by use of the NESTED procedure of the Genetic Data Analysis (GDA) program. Significance values were obtained using the methods of Li and Horwitz (1953) and Workman and Niswander (1970).
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Results |
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Allelic Frequencies
Two loci (Me-1 and G6pdh-1) were monomorphic in the four populations, and the other loci were polymorphic in at least one population (Table 1). Allele frequencies were heterogeneous between populations, and four alleles were exclusive to single populations: allele 2, locus Po-1, PEI population; allele 3, locus Acp-1, CNT population; allele 2, locus Skdh-1, FNI population; and allele 3, locus Est-1, PEI population.
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Genetic Diversity
The genetic diversities are presented in Table 2. The lower values of P are due to the presence of rare alleles in some populations or fixation of some alleles that are not fixed in other populations. The average of P was 77.8%, superior to the population's individual values. The value of A was an average of 2.0, being 1.6, 1.6, 1.3, and 1.4 in the populations CNT, PEI, FNI, and SPA, respectively, not differing from each other. The values of A for the populations FNI (1.3) and SPA (1.4) were different from the average (2.0).
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The values of Ho and He were 0.217 and 0.177, respectively, for the CNT population, 0.106 and 0.095 for the PEI population, 0.162 and 0.101 for the FNI population, and 0.085 and 0.136 for the SPA population. The averages were 0.148 and 0.187, respectively, for Ho and He, and they did not differ from each other within or among the populations.
Genetic Structure
Estimated values of the F-statistics are presented in Table 3. The results indicate a high divergence among the populations, with an average FSR of 0.380. In spite of the high divergence among the populations, the values of FRT were reduced (-0.069). Such results indicate that the divergence is not associated with the geographical distances, but rather is associated with the differences among the populations within regions.
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The average FIT was 0.275, which indicates a high total endogamy. The average FIS was -0.170, indicating an excess of heterozygotes in the population. Both these values suggest the effects of subdivision.
The estimates of the F-statistics for the two gaps in the CNT population are presented in Table 4. The results indicate a reduced divergence among the gaps, with an average FGT of 0.062. The average FIT was -0.152, indicating an excess of heterozygotes, consistent with the endogamy index. The average FIG was -0.228, and this indicates an excess of heterozygotes in the gaps. These results suggest that the subdivisional structure obtained by population analysis was not associated with the gap structure.
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Genetic Distances
The population groupings accomplished through the UPGMA technique are not consistent with the geographical distribution of the studied populations (Table 5 and Figure 1). The CNT population in the State of São Paulo is genetically closer to the FNI and SPA populations in the State of Santa Catarina, physically more than 350 km away, than the population of PEI, physically about 20 km from the sampled CNT population. The FNI population in the State of Santa Catarina is genetically closer to the CNT population in the State of São Paulo than it is to the population of SPA, at an approximate distance of 60 km. Such results are consistent with the F-statistics for these areas, indicating that differences among regions have smaller implications in the distribution of genetic variation.
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Discussion |
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The diversity indices found in P. cernuum are very high compared to the values found in other Piper species studied in Costa Rica: P. amalago, P. pseudo-fuligineum, and P. jacquemontianum (Heywood and Fleming 1986). The values of Ho were superior to He in three of the four studied populations, which indicates an excess of heterozygotes.
The average FIS of -0.170 also indicates an excess of heterozygotes in populations, suggesting a possible selection in their favor. It is possible to confirm this by progeny analysis, as accomplished by Reis et al. (2000) for Euterpe edulis (a neotropical understory palm), where the excess of heterozygotes in adults was not verified in the progenies.
The only population that did not present excess heterozygotes was the SPA population. This population was the only one located in an area of smaller forest covering, forming the smallest fragment in an area of many small fragments. The fragmentation could have accentuated the effects of genetic drift, causing the decrease in heterozygosity. An effect of forest fragmentation is to decrease population sizes, driven by genetic drift, thus extending inbreeding depression (Oyama 1993). These factors can have a negative influence on average individual fitness. The loss of genetic variability can diminish future adaptability to changing environments (Lande 1988). For example, fragmentation has reduced the reproductive success, through reductions in germination rate and fruit production, of Spondias mombin (Anacardiaceae), a early successional species that is intolerant of low light levels during recruitment (Nason and Hamrick 1997).
The divergence values among populations of P. cernuum are high, indicating a high structure for the species. Starr and Carthew (1998), studying the genetic structure of Hakea carinata (Proteaceae), a common shrub occurring in Australian forests, found a divergence of 46.9% among populations. Divergence has also been found among adult populations of Lisianthius skinneri, a tropical shrub occurring in Panama, presenting a divergence among populations of greater than 90%, due to, among other factors, founder events (Sytsma and Schaal 1985).
According to our analysis, although the excess of heterozygotes among gaps (FIG = -0.228) is maintained within them, the endogamy on a specific populational level disappears and the value of excess of heterozygotes (FIT = -0.152) remains. The value of FGT (0.062) is reduced, indicating a similarity among gaps. Such comparisons show that differences are among the populations, and are not applicable to groups of plants (gaps) within the same studied population.
The average FRT of -0.069 indicates a reduced divergence among regions, consistent with the similar allele frequencies among populations of different areas and with the different allele frequencies among populations of the same area. Also, the noncoherence among the geographical and genetic distances is demonstrably consistent with the lower value of FRT, indicating that the differences are not associated with the geographical variation.
However, in spite of the great displacement capacity of bats, flying several kilometers per night (Fleming et al. 1977; Shilton et al. 1999), such displacement takes place only if food is not readily available close to their day roost. In the case where food can be found near the day roost, bats do not fly great distances and remain within a restricted feeding area (Fleming and Heithaus 1981; Hamrick and Loveless 1986). In the case of the PEI and CNT populations, which are continuous and well-preserved areas, the lack of food should not be a decisive factor for the displacement of bats to other areas. This would reduce the gene flow among the areas and would also explain the high divergence among them.
Hamrick and Loveless (1986) verified that, unexpectedly, animal-dispersed species had higher divergence among populations, indicating that the majority of the seeds ingested or attached are deposited locally and that long-distance dispersal routinely leads to the establishment of new populations by one or a very few individuals, suggesting relatively little gene flow among established populations. Moreover, in spite of the fact that seeds may not necessarily be taken long distances, they may be clump dispersed. The bats eat below the night roost, altering individual seed distribution and genotype (Hamrick and Loveless 1986).
On other hand, pollination mechanisms are one of the first factors to determine gene flow levels in plant populations (Govindaraju 1988). A smaller divergence among populations may occur in the case where pollinators had long flights. However, this does not seem to be the case in the Piperaceae species, where pollination is accomplished by the wind, small bees, wasps, and flies (Heithaus 1979; Semple 1974).
Founder events can be greatly responsible for the structuring of natural populations of P. cernuum, as in L. skinneri (Sytsma and Schaal 1985), and consequently are responsible for the great divergence among populations, as among the geographically closest populations. Locus Pgdh-1 is an example of how the founder events are able to structure the natural populations of P. cernuum. Locus Pgdh-1 presents a high frequency of allele 1 in the CNT and SPA populations, reaching fixation in the FNI population, while allele 2 is almost fixed in the PEI population. Several other alleles are shown to be exclusive in certain populations (Table 1), reinforcing the hypothesis that founder events have a decisive role in the formed structure.
Thus the establishment of small populations (few individuals) in areas previously not occupied by the species cannot increase the divergences among the populations. A single seed can colonize a gap, and when it reaches its reproductive state, it can be responsible for the occupation of other gaps that are being formed nearby. This aspect is reinforced by the lower divergence among the gaps analyzed. Thus P. cernuum possesses a high divergence among populations, seemingly caused by the effect associated with founder gap dynamics.
Consequently, in P. cernuum conservation and management it is necessary to maintain reproductive branches and dispersers, since the seeds produced by the existing populations will be responsible for the occupation of new gaps and new population foundations, thus maintaining the dynamics of allele movement in this species and making available new individuals for new exploitation cycles. More studies on recruitment and pollination are also necessary to better understand conservation and management strategies in P. cernuum.
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Acknowledgments |
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The authors thank Gilberto Dalagnol for his invaluable help in the electrophoresis work. This research was supported by FNMA, CAPES, and Universidade Federal de Santa Catarina.
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Footnotes |
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Corresponding Editor: J. Perry Gustafson
Received January 22, 2002
Accepted September 5, 2002
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