Genetic Structure of a Mimosoid Tree Deprived ... - Wiley Online Library

Genetic Structure of a Mimosoid Tree Deprived of Its Seed Disperser, the Spider Monkey LUIS F. PACHECO* AND JAVIER A. SIMONETTI Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile

Abstract: To assess the genetic consequences for a Neotropical tree of the loss of its main seed disperser, we compared the genetic structure of Inga ingoides in a site where the spider monkey (Ateles paniscus) was abundant and a site where it had been eliminated by subsistence hunting. Gene flow should be reduced in the site where the spider monkey is absent, and there should be a corresponding subpopulation differentiation of seedlings within the spatial range of the movements of these primates in the absence of between-site differences in allelic frequencies. At the microhabitat ( family) scale, seedlings growing under parent plants should be genetically more related in the absence of the spider monkey than in its presence. Subpopulation differentiation was smaller where the spider monkey was present ( four loci, FST ⫽ 0.011) than where it was absent ( four loci, FST ⫽ 0.053) for the first year of study, but not for the second year (three loci, FST ⫽ 0.005 vs. 0.003). The number of alleles in common among seedlings growing under parent plants was smaller in the presence of the spider monkey than in its absence, showing family genetic structure in the first generation for both years of study (MannWhitney, z ⫽ ⫺2.17, p ⫽ 0.03 and z ⫽ ⫺2.72, p ⫽ 0.006 for 1996 and 1997, respectively). This family genetic structure in the first generation should accelerate the development of population genetic structure. Development of genetic structure might result in demographic changes, one of which would be a fitness reduction if the species were self-incompatible, as suggested for Inga by available evidence. Large birds and mammals are the main targets of subsistence hunting in the Neotropics. Extinction of seed-dispersing frugivores may result in pronounced changes in the demographic and genetic structure of tree species in Neotropical forests. Estructura Genética de un Arbol Mimósido Privado de su Dispersor de Semillas, el Marimono Resumen: Para evaluar las consecuencias genéticas para una especie de árbol neotropical de la pérdida de su principal dispersor de semillas, comparamos la estructura genética de Inga ingoides entre un sitio con abundancia de marimono (Ateles paniscus) y otro donde éste había sido eliminado por cacería de subsistencia, en la estación Biológica Beni, Bolivia. El flujo génico debería estar reducido en ausencia del marimono, con una correspondiente diferenciación de subpoblaciones de plántulas dentro de la escala espacial de movimiento de dichos primates, si las frecuencias alélicas no difieren entre sitios. A escala familiar, las plántulas que crecen debajo de los parentales deberían estar más emparentadas entre sí en ausencia del marimono que en su presencia. La diferenciación de subpoblaciones fue mayor en ausencia del marimono (cuatro loci, FST ⫽ 0.053) que en su presencia (cuatro loci, FST ⫽ 0.011) para el primer año de muestreo, pero no así para el segundo año (tres loci, FST ⫽ 0.003 vs FST ⫽ 0.005). El número de alelos comunes para plántulas que crecen debajo de parentales fue mayor en ausencia del marimono, que en su presencia, evidenciando la formación de una estructura genética familiar en la primera generación para ambos años de muestreo (MannWhitney, z ⫽ ⫺2.17, p ⫽ 0.03, y z ⫽ ⫺2.72, p ⫽ 0.006 para 1996 y 1997, respectivamente). Esto aceleraría el proceso de estructuración genética a nivel poblacional. El desarrollo de estructura genética podría producir cambios demográficos, uno de los cuales sería la reducción en la adecuación biológica, si la especie es autoincompatible como lo sugiere la evidencia existente sobre Inga. La cacería de subsistencia se enfoca en los mamíferos y aves de gran tamaño. La extinción de frugívoros dispersores de semillas podría generar cambios grandes en la estructuración demográfica y genética en los bosques tropicales.

* Current address: Estación Biológica Tunquini, Instituto de Ecología, Casilla 10077, Universidad Mayor de San Andrés, La Paz, Bolivia, email [email protected] Paper submitted April 7, 1999; revised manuscript accepted February 23, 2000.

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Introduction Seed dispersal conveys several advantages for plants, such as colonization opportunities, reduced competition between both related seedlings and parent and offspring, and escape from parental-related mortality and mating between close relatives (Dirzo & Domínguez 1986). Loss of seed dispersers is associated with reduced seedling density and diversity (Chapman & Onderdonk 1998). The extinction of dispersal agents is also associated with a reduction in the number of seeds that disperse long distances (⬎20 m) from parental trees and probably with a reduction in the total number of germinated seeds (Pacheco & Simonetti 1998). As a result, populations of the affected species might become less dense and spatially more aggregated in the long term. Furthermore, seed dispersal is a mechanism of gene flow (Antonovics 1968); therefore, the loss of seed dispersers will affect not only demographic and spatial patterns but also gene-flow patterns and hence population genetic structure. Tropical species that are biotically dispersed generally show less population differentiation than those abiotically dispersed (Loveless 1992). Gene flow reduces the probability of developing genetic structure, which could lead to the formation of family groups, population differentiation, and subsequent increase in inbreeding (Antonovics 1968; Allendorf 1983; Liu & Godt 1983; Loveless & Hamrick 1984; Lande 1988; Slatkin 1989, 1993; Schnabel & Hamrick 1990; Hamrick et al. 1991, 1993). The loss of the seed-dispersal agent might trigger changes in the genetic structure within a population. Local species extinction is a common consequence of human activities. Rural human populations in the Neotropics depend largely on wildlife to meet their nutritional needs (Redford & Robinson 1987). Subsistence hunting in the Neotropics focuses on large herbivorous birds and mammals, and local extinction is a frequent outcome (i.e., Peres 1990; García & Tarifa 1991). Extreme cases result in an “empty forest,” a forest apparently intact but actually altered in its plant-animal interactions (Redford 1992). In this regard, subsistence hunting could be a hidden factor shaping the genetic structure of vertebrate-dispersed species. Although the effects of seed dispersal on the genetic structure of tropical trees have been explored (e.g., Hamrick & Nason 1996; Nason et al. 1997), the relationship between a human-induced extinction and its effects on genetic diversity has not been considered. Hamrick et al. (1993) found that, for three tropical tree species, near neighbors (within the small and intermediate size classes) have more alleles in common than randomly chosen individuals. They hypothesized that spatially clustered individuals have at least one parent in common. Taking this prediction further, the absence of seed dispersal results in all seedlings growing under pa-

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rental canopies being at least half siblings, whereas seed dispersal acts to mix the offspring of several individuals. Therefore, seedlings growing under parental canopies in the presence of seed dispersal include some individuals that are not offspring of the tree under which they are growing. We evaluated this prediction for a Neotropical tree following the loss of its main seed disperser to subsistence hunting in an otherwise “intact” forest at the Biosphere Reserve Estación Biológica Beni (EBB) in Bolivia. Inga ingoides (Mimosoideae, locally known as pacay cola de mono) is a hermaphroditic tree common at the EBB (Candolle 1993). Its seeds are dispersed almost exclusively by the spider monkey (Ateles paniscus; Cebidae, locally known as marimono; Pacheco & Simonetti 1998). Given that spider monkeys can move over 1 km/day (Symington 1988), seed dispersal should prevent population differentiation within this spatial range. In the absence of spider monkeys, however, post-fertilization gene flow (via seeds) may be eliminated, which could trigger the development of genetic structure in populations of I. ingoides. Although genetic structure at the population level (differentiation of subpopulations) may not be evident in the first generation (a cohort), it will be at the family scale (seedlings under parental canopies). In the absence of post-fertilization gene flow, I. ingoides seedlings under parents will all be at least half siblings, assuming that the species is not self-compatible. In areas where the spider monkey is present, some seedlings will not be offspring of the tree under which they are growing because they emerge from dispersed seeds.

Study Area and Methods The Biosphere Reserve Estación Biológica Beni, Bolivia (lat 14⬚30⬘–14⬚50⬘S, long 66⬚00⬘–66⬚40⬘W), covers 135,000 ha of lowland forests mixed with savannas and wetlands. A recent review reported 451 species of vascular plants (Roldán et al. 1999), although over 2000 species are estimated to occur at the EBB (Miranda et al. 1991). Most important families by number of species are Fabaceae, Rubiaceae, Euphorbiaceae, and Asteraceae ( Roldán et al. 1999). Besides the spider monkey, four other species of primates occur at EBB: howler monkey (Alouatta seniculus), capuchin monkey (Cebus apella), squirrel monkey (Saimiri boliviensis), and night monkey (Aoutus azarae) (García & Tarifa 1991). All of them are potential I. ingoides consumers, but the spider monkey may be the main if not the only one that ingests whole seeds (Pacheco & Simonetti 1998). The two study sites were within the same continuous forest but were separated by about 30 km. The site where the spider monkey was eliminated by subsistence hunting (disperser absent, DA) is approximately 2 km

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north of a Tsimane Indian settlement called “08.” This settlement is ⬎30 years old and the spider monkey has been locally extinct for about 10 years. The other site, Campo Mono (disperser present, DP), harbors large populations of spider monkeys and other large mammals (García & Tarifa 1991; Roldán et al. 2000). Vegetation in both sites corresponds to seasonally flooded tropical alluvial forest that is inundated 3 or 4 months a year ( Miranda et al. 1991). Study sites were, however, only partially flooded. In both sites, forests had an emergent stratum (⬎40 m high), two arboreal strata (30 and 20 m), and one stratum of shrubs and small trees (5 m). Sites were floristically similar in tree species composition. The most abundant tree species at both sites were Astrocaryum gratum, Rheedia spp., Pseudolmedia laevis, Trichilia spp., Celtis schippi, Virola sebifera, and Chamaedorea angustisecta (Roldán 1997). Field observations of a group of spider monkeys (DP site) helped us define two zones separated by 1 km that were visited frequently by the same monkey group. Given that spider monkeys can travel 1 km in a few minutes and that seed passage through its digestive tract takes more than 4 hours (Pacheco & Simonetti 1998), we considered 1 km a likely distance of dispersal for I. ingoides. Based on these considerations, we operationally defined subpopulations as the group of trees growing in those two zones (DP1 and DP2). Two groups of I. ingoides trees separated by 1 km were operationally defined as subpopulations (DA1 and DA2) at the other site. In this context, we compared the genetic structure of I. ingoides at three scales. First, we compared individuals from the area where the spider monkey was present (DP) to individuals from the area where it had been extirpated by hunting (DA). The seed-dispersal processes affecting both populations of adults must have been similar; so we did not expect to find between-site differences in the genetic structure. Second, within those areas, and under the assumption of no difference in allele frequencies between the areas, we compared the differentiation between the subpopulations from each site. At this level, genetic differentiation of seedlings should be greater between DA1 and DA2 than between DP1 and DP2, if the absence of the spider monkey has an effect and if there are no between-site differences in allelic frequencies. Third, we compared the amount of genetic information shared by seedlings within groups collected immediately under adult I. ingoides trees, (hereafter called “undercanopy groups”). Here, similarity should be greater within undercanopy groups from DA than within those from DP, under the same assumption. Therefore, the amount of genetic information shared by seedlings should decrease as the number of undercanopy groups considered as one group of seedlings increases from one to two and so on (Fig. 1). We chose 10 adult trees (focal individuals) within each


Pacheco & Simonetti

Figure 1. Hypothetical model for variation of the amount of genetic information shared by groups of Inga ingoides seedlings collected under parents (undercanopy groups), as a function of the number of undercanopy groups considered as a group of seedlings for a site where the dispersal agent is present (DP) and another where the dispersal agent is absent (DA).

subpopulation and took leaf and bark samples from each to be electrophoretically analyzed. Under each focal tree and within a radius equal to its canopy, 20 seedlings (root included) were taken as representatives of the undercanopy groups. Samples were collected twice within the same subpopulations, but not from the same focal trees, in August 1996 and April 1997 and were kept on ice until enzyme extraction, which was done at the Universidad de Chile, Santiago, between 3 and 10 days after field collection. We analyzed 14 enzyme systems. The extraction buffer was tris 0.05M; citrate 0.15%; cysteine ClH 0.1%; Na l-ascorbate 0.1%; Polyethylen-glycol 1% (molecular weight 15,000–20,000); soluble PVP (high molecular weight 360,000); Mercapto-ethanol, 2 drops/100 mL (D. Sepúlveda, personal communication). Centrifuged extracts were stored first at ⫺70⬚ C and later at ⫺20⬚ C until electrophoresis. For electrophoresis, extracts were adsorbed with 1.5 ⫻ 12 mm paper wicks and run in three buffer media: buffers A and D (Conkle et al. 1982) and buffer H (“Histidine-citrate discontinuous;” Murphy et al. 1990). General electrophoretic and staining procedures were based on those of Conkle et al. (1982). Some modified staining procedures were assayed following Wendel and Weeden (1989). Staining for the only two enzyme systems that were finally used for analysis (peroxidase, PRX, and phosphoglucose-isomerase [PGI]) was based entirely on the technique of Conkle et al. (1982) and run in buffer A. Only these systems were used because of their consistent interpretation from gels and because the partial information given by the other systems was not adequate for analysis.

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Testing the Assumption of No Difference in Allele Frequencies between Sites To avoid taking differences between sites in support of the hypothesis of an effect of the absence of seed dispersal which may only reflect differences in allele frequencies, we compared the heterozygosity between the two populations. Heterozygosity was calculated for each locus for each population and compared with a MannWhitney U test (Zar 1984). Post-Fertilization Gene Flow To evaluate whether the absence of the spider monkey affected post-fertilization gene flow of I. ingoides at DA site in relation to DP, we compared the genotypes for each locus and seedling analyzed to the genotypes of the adults under which seedlings were collected. We considered those seedlings not sharing at least one allele at each locus with the adult trees under which they were growing as the product of seed dispersal (immigrants) and as representing post-fertilization gene flow. The binomial proportion of trees under which immigrants were found was compared between sites with Fisher’s exact test (Zar 1984). This estimate of gene flow cannot differentiate within- from among-subpopulation gene flow, but it may show the effect of the seed disperser. Also, immigrants that share alleles with the tree under which they were growing could not be identified by this method, but this only makes our estimate of the potential effect of seed dispersal more conservative. Gene flow as defined here has no direct relationship with the most common Nm estimator, which includes pre-fertilization gene flow. Genetic Structure We calculated Wright’s F statistics FIS and FST (Weir & Cockerham 1984) for the first spatial scale and over all cohorts (adults, saplings, and seedlings) corresponding to all individuals collected at each site (DP vs. DA). We present these only as basic information for I. ingoides. To evaluate the development of genetic structure in I. ingoides seedling populations as a consequence of the extinction of the spider monkey, we analyzed data considering the other two spatial scales. At the second scale, subpopulations within each site ( DP1 and DP2, DA1 and DA2), we calculated FIS and FST (according to Weir & Cockerham 1984), and exact tests of genic and genotypic differentiation for each pair of subpopulations within each site based on GENEPOP, version 1.2 (Raymond & Rousset 1995a, 1995b). The chi-square tests proposed by Li and Horvitz (1953) and Workman and Niswander (1970) (both cited by Godt & Hamrick 1993; Gibson & Wheelwright 1995)


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were used to test for significant deviations from zero for the FIS and FST (single-locus estimates). The multilocus FST estimates were considered significant when the multilocus genic and/or genotypic differentiation tests showed significant differences. When FIS or FST were significantly different from zero, we estimated their variances using “jackknife” procedures (Eguiarte et al. 1992). One individual at a time was excluded for the calculation of each pseudovalue for subpopulations of focal trees, and one undercanopy group was excluded for the calculation of each pseudovalue in the case of seedling subpopulations. We used information for subpopulations of adults for comparison with seedling subpopulations. To evaluate development of family genetic structure, we used the third scale of analysis, which consisted of counting the number of alleles in common (NAC; Surles et al. 1990; modified by Hamrick et al. 1993) for seedlings in each undercanopy group. Within each undercanopy group (the 20 seedlings under each focal tree), one seedling (i ) at a time was taken and compared to another randomly chosen individual ( j ) from the same undercanopy group. An NAC observation was obtained per locus for each individual. We summed these observations over loci (from locus 1 to locus k), over individuals (from individual 1 to individual n), within each undercanopy group (from undercanopy group 1 to p), and for both subpopulations at each site (from subpopulation 1 to s, where s ⫽ 2) to obtain a grand mean and variance based on the total N individual NAC observations for groups of seedlings, considering each undercanopy group as one group. The mean therefore equals s

p n k

1

1

NAC = 1 ⁄ N ∑ ∑ ∑ ∑ NAC ij . 1

(1)

1

Undercanopy groups were then randomly pooled in pairs (within the same subpopulation), and the procedure was repeated to obtain a new NAC observation for each individual. We summed these new NAC observations according to equation 1 to obtain a grand mean and variance for groups of seedlings, including two undercanopy groups ( p was reduced to the total resulting from the pooling of undercanopy groups in pairs). For those cases when pooling left single undercanopy groups, these pairs were completed with one of the undercanopy groups already considered in another group. Some of the NAC observations were the same as when undercanopy groups were considered singly in cases when the comparison involved the same two individuals, or when the new, randomly chosen individual had the same genotype as the one chosen before. This produced a potential statistical dependence of data, which limited statistical analysis. The procedure of pooling undercanopy groups and obtaining new NAC observations ended when the groups contained all undercanopy groups in a subpopulation.


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Table 1.

Electrophoretic results for Inga ingoides collected at the Estación Biológica Beni, Bolivia.

Enzyme AAP AAT ACP CAT FL-EST IDH LAP MDH MNR 6-PGD PGI PGM PRX TPI Total

Pacheco & Simonetti

No. of loci

Polymorphic loci

Alleles/locus

? 2 2 1 ? ? ? 1 1 2 1 1 5 1 17

? 2 2 1 ? ? ? 1 1 0 1 0 3 0 11

? 2?, 2? 2, 2 2 ? ? ? 2 3? 1, 1 3 1 4, 4, 4, 2, 1 1 2.1 ⫾ 0.31*

Alleles/polymorphic locus ?

Resolution no partial partial partial no no no partial partial partial good partial good partial

2, 2 2 ? ? ? 2 3 4, 4, 4 2.9 ⫾ 0.35*

*Means ⫾ SE (only for those enzymes adequately characterized).

For statistical analysis, NAC observations with undercanopy groups were considered singly, and NAC observations with all undercanopy groups as one pooled group of seedlings were compared between sites. The number of undercanopy groups considered in the last group differed between years, depending on the sample size. Both comparisons were done with a Mann-Whitney test. We illustrated the general tendency by plotting mean NAC versus number of undercanopy groups considered in one group, and the ratio of NAC for each point (according to the number of undercanopy groups considered) to the NAC when all undercanopy groups were considered as one group of seedlings was used to describe genetic structure at the family scale (Hamrick et al. 1993).

Results Nine of the 14 enzyme systems we assayed resolved at least partially, but only two were used for analysis: PRX, three loci in 1996 and two loci in 1997 (four alleles/locus, all loci), and PGI, one locus (three alleles) in both years. Eleven of 17 loci were polymorphic (following Hartl 1990) (Table 1). Only those individuals that could

be clearly interpreted from gels were used for analysis, which resulted in a reduction of original sample sizes, as shown in the corresponding tables. No differences in allele frequencies were found between the two sites for either parental or seedling populations for any of the 2 years of sampling (Table 2). Post-Fertilization Gene Flow Post-fertilization gene flow was greater in the presence of the spider monkey (DP site) for both years. In 1996, 28 ⫾ 6% (mean ⫾ SE) of the seedlings from DP undercanopy groups were not offspring of the adult tree under which they were growing. These immigrants were present in 85% (11 out of 13) of the undercanopy groups from DP, whereas none of the nine undercanopy groups from DA included immigrants in 1996. In 1997, differences followed the same pattern but were not as clear. Immigrants occurred in 53% (9 out of 17) of DP undercanopy groups and in only 29% (5 out of 17) of DA undercanopy groups, although no statistical difference between sites was found (Fisher’s exact test, p ⫽ 0.148). The mean number of immigrants per undercanopy group was twice as high in DP than in DA groups (12 ⫾ 4% vs. 6 ⫾ 3%) in 1997, but the difference

Table 2. Comparison of heterozygosity (Ho) between populations of Inga ingoides at two sites and two life stages at the Estación Biológica Beni, Bolivia.*

Life stage and year (n) Trees 1996 (4) Trees 1997 (3) Seedlings 1996 (4) Seedlings 1997 (3)

Ho at disperserpresent site

U

p

0.62 (0.25 – 0.89) 0.41 (0.12 – 0.75) 0.38 (0.06 – 0.61) 0.27 (0.08 – 0.49)

0.44 (0.18 – 0.69) 0.52 (0.18 – 0.81) 0.36 (0.14 – 0.49) 0.34 (0.10 – 0.63)

5 3 7 3

⬎0.2 ⬎0.2 ⬎0.2 ⬎0.2

*Figures for Ho are mean and range. The number of loci used for analysis each year is indicated by n.


Mann Whitney

Ho at disperserabsent site

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Table 3. Genetic structure estimates for Inga ingoides based on two populations of adults and seedlings from Estación Biológica Beni, Bolivia. Locusb Year and structure measurea 1996 FIS FST p genic p genotypic 1997 FIS FST p genic p genotypic

PGI

PRX1

PRX2

PRX3 Multilocus

Adult 19, 34

Seedling 190, 502

Adult 22, —

Seedling 207, —

Adult 22, 32

Seedling 208, 386

Adult 22, 32

Seedling 211, 389

Adult

Seedling

⫺0.033 ⫺0.050 0.999 0.999

⫺0.044 0.007 0.214 0.185

0.160 ⫺0.046 0.947 0.941

0.059 0.007* 0.046 0.046

⫺0.141 ⫺0.016 0.634 0.375

0.146* 0.023* ⬍0.001 ⬍0.001

⫺0.251 0.046 0.090 0.054

⫺0.067 0.029* ⬍0.001 ⬍0.001

⫺0.068 ⫺0.009 0.647 0.441

0.034 0.019 ⬍0.001 ⬍0.001

— — — —

— — — —

⫺0.169 ⫺0.009 0.643 0.591

0.105* 0.008* 0.109 0.188

⫺0.192 ⫺ 0.001 0.397 0.305

⫺0.067 0.007* 0.020 0.016

⫺0.167 ⫺0.005 0.677 0.619

0.005 0.006 0.014 0.018

⫺0.046 ⫺0.010 0.617 0.600

0.100* 0.000 0.128 0.156

a The FIS and FST ( Weir & Cockerham 1984) values significantly different from zero ( by ␹ 2 tests) are marked with asterisks: *p ⬍ 0.05 and **p ⬍ 0.01. Probabilities for genic and genotypic differentiation tests ( Raymond & Rousset 1995a, 1995b) are shown as additional measures of genetic structure. b Numbers below the locus name are sample sizes for 1996 and 1997, respectively. There are no data for locus PRX1 for 1997.

ling subpopulations at that site (multilocus FST ⫽ 0.011; Table 4). No significant difference between parental subpopulations was found for the DA site, whereas the considerable difference between subpopulations of seedlings (multilocus FST ⫽ 0.053) was supported by genic and genotypic differentiation tests (Table 4). Multilocus FST among seedling subpopulations at DP sites (mean from pseudovalues ⫽ 0.012; 95% CI [0.0151– 0.0281]) was smaller than that from DA sites (0.0694 [0.0558–0.0829]; t ⫽ 7.75; df ⫽ 22; p ⬍ 0.0001). Differentiation between seedling subpopulations at DA sites may not be explained by differences between parental subpopulations, as it could be for DP (Table 4). No subpopulation differentiation for parentals was found for either DA or DP sites in 1997. Multilocus FST be-

between both groups was not significant (Mann-Whitney, z ⫽ 1.34; p ⫽ 0.08). Genetic Structure At the scale of sites (DP vs. DA), none of the estimates of genetic structure were significantly different from zero for the parental populations (Table 3). In the case of seedlings, small (sensu Hartl 1990) but significant differentiation between sites was found for both years (Table 3). In 1996, a considerable (sensu Hartl 1990) differentiation was found between parental subpopulations from the DP site, although this differentiation was not detected by the genic or the genotypic differentiation tests. This differentiation was much reduced between seed-

Table 4. Subpopulation genetic structure for Inga ingoides for adults and seedlings in two sites (two subpopulations per site) at the Estación Biológica Beni, Bolivia in 1996. Locusb PGI Site and structure measure a Disperser present FIS FST p genic p genotypic Disperser absent FIS FST p genic p genotypic

PRX1

Adult 11, 8

Seedling 106, 83

Adult 13, 9

⫺0.043 0.031 0.999 0.999

⫺0.064 0.019* 0.172 0.135

0.283 0.214** 0.028 0.078

0.000 ⫺0.067 0.999 0.600

⫺0.029 0.018 0.084 0.085

⫺0.186 0.002 0.635 0.490

PRX2 Seedling 117, 90

PRX3

Seedling 117, 89

Adult 13, 9

Adult 13, 9

Seedling 120, 90

0.075 0.021** 0.025 0.033

⫺0.047 ⫺0.021 0.598 0.533

0.189 0.008 0.089 0.162

⫺0.044 ⫺0.048 0.898 0.879

⫺0.010 0.004 0.101 0.072

⫺0.026 0.068** ⬍0.001 ⬍0.001

⫺0.256 ⫺0.031 0.781 0.718

0.029 0.045** 0.024 0.020

⫺0.504 0.038 0.538 0.287

⫺0.159 0.045** ⬍0.001 0.001

Multilocus Adult

Seedling

0.048 0.058 0.433 0.577

0.073 0.011 0.010 0.011

⫺0.284 ⫺0.003 0.934 0.801

⫺0.062 0.053 ⬍0.001 ⬍0.001

a The FIS and FST ( Weir & Cockerham 1984) values significantly different from zero ( by ␹ 2 tests) are marked with asterisks: *p ⬍ 0.05 and **p ⬍ 0.01. Probabilities for genic and genotypic differentiation tests ( Raymond & Rousset 1995a, 1995b) are shown as additional measures of genetic structure. b Numbers below the locus name are sample sizes for 1996 and 1997, respectively.


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Table 5. Subpopulation genetic structure for Inga ingoides for adults and seedlings in two sites (two subpopulations per site) at the Estación Biológica Beni, Bolivia in 1997. Locusb Site and structure measurea Disperser present FIS FST p genic p genotypic Disperser absent FIS FST p genic p genotypic

PGI

PRX2

PRX3 Multilocus

Adult 17, 17

Seedling 259, 243

Adult 16, 16

Seedling 196, 190

Adult 16, 16

Seedling 197, 192

Adult

Seedling

⫺0.043 ⫺0.044 0.999 0.999

0.187** 0.011** 0.031 0.051

⫺0.297 ⫺0.021 0.831 0.780

0.116 ⫺0.003 0.174 0.294

⫺0.123 ⫺0.042 0.953 0.866

⫺0.110 0.009* 0.215 0.179

⫺0.169 ⫺0.035 0.999 0.992

⫺0.007 0.005 0.030 0.066

⫺0.011 0.011 0.433 0.668

0.092 0.004 0.132 0.092

⫺0.233 ⫺0.021 0.858 0.741

⫺0.025 0.004 0.409 0.381

⫺0.133 ⫺0.011 0.809 0.900

0.015 0.003 0.321 0.241

⫺0.002 ⫺0.027 0.653 0.670

0.011 ⫺0.003 0.530 0.532

a The FIS and FST ( Weir & Cockerham 1984) values significantly different from zero ( by ␹ 2 tests) are marked with asterisks *p ⬍ 0.05 and **p ⬍ 0.01. Probabilities for genic and genotypic differentiation tests (Raymond & Rousset 1995a, 1995b) are shown as additional measures of genetic structure. b Numbers below the locus name are sample sizes for 1996 and 1997, respectively.

tween seedling subpopulations at DP sites was small, but was evident in the genic differentiation test (Table 5). Multilocus FST for DP site (0.005 [0.004–0.007]), however, was not different from that of DA sites (0.004 [0.001–0.008]). Family genetic structure showed similar patterns for both years. The NAC considering each undercanopy group as a single group of seedlings was larger for DA than for DP in both years ( Mann-Whitney, z ⫽ ⫺2.17, p ⫽ 0.03 and z ⫽ ⫺2.72, p ⫽ 0.006 for 1996 and 1997, respectively). This indicates that seedlings collected under parental canopies were more related in the DA site than those collected under adults in the DP site. This difference tended to disappear as more undercanopy groups were considered (Fig. 2). For the DP site, there was little difference between mean NAC when undercanopy groups were considered singly or all in one group (NAC1/NAC6 ⫽ 1.061 for 1996; NAC1/NAC9 ⫽ 1.046 for 1997), although that difference was greater for the DA site in both years (NAC1/ NAC5 ⫽ 1.218 for 1996 and NAC1/NAC9 ⫽ 1.088 for 1997). For 1996 there were no significant differences between sites for NAC5 (five undercanopy groups in one group; Mann-Whitney z ⫽ ⫺0.56, p ⫽ 0.57) or for 1997 for NAC9 (z ⫽ ⫺0.84; p ⫽ 0.39). The effect of the seed disperser at this scale was to produce undercanopy groups formed by seedlings with a degree of relatedness similar to that within subpopulations in DP (no statistical test is presented for this assertion because of the potential statistical dependence of observations).

Discussion The disappearance of seed dispersers such as primates may result in changes in populations of dispersed spe-


Figure 2. Genetic information shared (number of alleles in common, mean ⫾ SE) by groups of Inga ingoides seedlings collected under adult trees (undercanopy groups), as a function of the number of undercanopy groups considered as a group of seedlings, for a site where spider monkey is present (DP) and another where it is absent (DA): (a) 1996 and (b) 1997.

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cies. Local extinction of primates might lead to reduced germination, which affects seedling densities and species richness at the seedling stage (Chapman & Chapman 1995; Chapman & Onderdonk 1998) and may modify the seed shadow and spatial seedling recruitment pattern of seedlings (Pacheco & Simonetti 1998). Our results strongly suggest that for Inga-Ateles, in addition to the spatial and demographic attributes of the trees, their genetic structure might also be affected by disappearance of a seed disperser. Post-fertilization gene flow of I. ingoides appears to be reduced in the absence of the spider monkey. The fact that post-fertilization gene flow was not significantly reduced in 1997, as it was in 1996, may be explained by the conservative way we defined immigrants. Seeds transported by dispersers, but sharing at least one allele per locus with the trees under which they were defecated and germinated, could not be distinguished (by our method) from seeds falling and germinating under their parent tree. We believe that our method greatly underestimated post-fertilization gene flow. Supporting this view, family genetic structure was evident in seedling populations for both years of sampling. Therefore, seed dispersal by the spider monkey seems to reduce the aggregation of related seedlings compared to what occurs when those primates are absent. Genetic structure is likely to persist through the adult stage, although it may become less evident, as in Cecropia obtusifolia (Alvarez-Buylla et al. 1996). The fact that post-fertilization gene flow in the absence of the spider monkey is not zero should only retard the development of genetic structure in adult populations of I. ingoides. Gene flow tends to differ from year to year (Hamrick 1982), and temporal variability in gene flow was evident in this study. In the absence of the spider monkey, the development of subpopulation genetic structure (measured by FST) was suggested in 1996 but not in 1997 (Tables 4 & 5). Accordingly, our estimates of post-fertilization gene flow were zero in the absence of that primate only for 1996. Also, seedlings of I. ingoides occurring away from parental trees (⬎20 m) were much more common in 1997 than in 1996 for the same study area from which spider monkeys were absent (Pacheco & Simonetti 1998). Even a low rate of long-distance gene flow may preclude population differentiation in absence of selection (Allendorf 1983; Loveless & Hamrick 1984), which may explain the different patterns of genetic structure between years in this study. An alternative but not exclusive explanation is that one generation (a seedling cohort) may be too little time for changes at the subpopulation level to be detected, especially if gene flow via pollen is considerable (Hamrick & Nason 1996). This may be the case for I. ingoides, given that its pollination may be similar to that of other Inga species, which are pollinated by agents as diverse and mobile as hummingbirds, insects, and bats (Koptur 1983, 1984). In any


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event, development of family genetic structure, which is clear for both years of study in the absence of the spider monkey, should accelerate the process of genetic structuring at the subpopulation level in a few generations. Dispersal in populations of I. ingoides, where the spider monkey is absent, now occurs almost entirely by gravity, which should trigger population differentiation and increase inbreeding (Loveless & Hamrick 1984; Hamrick & Loveless 1986; Slatkin 1987; Hamrick & Nason 1996; Nason et al. 1997). It is possible to predict that family genetic structure will in turn affect the demography of I. ingoides in the long term. Inbreeding in plants occurs in two ways, selfing and mating between close relatives, and occurs more frequently in small populations or when spatial genetic structure is present (Ellstrand & Elam 1993). Koptur (1984) found that fruit production in Inga is greater when fertilization occurs with pollen from a distant tree than when it occurs with pollen from a near neighbor, and it is greater in both cases than that from selfing. She also found that crosses between distant trees were more effective than open (natural) pollination, showing that the latter involves a significant proportion of matings between neighbors and relatives, which may be a general pattern (Hamrick & Murawski 1990; Hamrick & Nason 1996). For I. ingoides, the absence of the spider monkey increases the probability of a near neighbor being a close relative. Therefore, if I. ingoides is self-incompatible, its fitness will be negatively affected (reduction in pollination efficiency) by development of family genetic structure. If, in contrast, I. ingoides is self-compatible (totally or partially), then family genetic structure will accelerate the differentiation of subpopulations (Bawa 1990). Finally, continuous mating among relatives will increase inbreeding, probably leading to a depression in evolutionary possibilities and consequent secondary effects (Loveless & Hamrick 1984; Foster-Huenneke 1991; Ellstrand & Elam 1993). Inbreeding allows expression of deleterious genes and promotes loss of heterozygosity (Chambers 1983). Inbreeding depression will be more pronounced in typically out-crossing species than in those that normally reproduce by selfing (Ellstrand & Elam 1993). Inga ingoides may be self-incompatible, as were all six species of Inga studied by Koptur (1984). Thus, an increase in inbreeding forced by genetic structure may be especially negative for the fitness of the population. Furthermore, the extinction of the spider monkey may also have indirect effects on the demography of I. ingoides. As an example, Coley and Barone (1996) suggest that distance-limited dispersal for several generations may promote the development of more virulent pathogens that are better adapted to the parental genotype. Therefore, offspring genetically differentiated from parentals will be favored. In general, the spatial distribution of genetic variability may significantly affect the evolutionary


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dynamics of populations ( Wright 1982; Linhart & Grant 1996). Even when the scale of genetic structure is fine, this may promote the formation of new genotypes locally. At sites where the absence of the spider monkey continues, at least two consequences may follow: direct negative effects on the demography of I. ingoides and limitations of its evolutionary possibilities. The effects of loss of the spider monkey could be extended to other species as well. At least 29 species of fruit are eaten by the spider monkeys at EBB (F. Méndez, unpublished data), and this primate is considered the only seed disperser of at least 11 species in Surinam (van Roosmalen 1985a, 1985b). Furthermore, existing evidence suggests that subsistence hunting tends to be unsustainable and that defaunation will be more severe in the future (Robinson & Bodmer 1999). If we consider that nearly 90% of mature forest species are dispersed by animals (van Roosmalen 1985a, 1985b), it is easy to imagine that forest demographic and genetic structures will change enormously if defaunation continues to increase. In this way, subsistence hunting may modify the diversity of tropical forests by changing the genetic structure of plant populations through the local or functional extinction of primates.

Acknowledgments Field and lab work were supported by grants from Man and the Biosphere United Nations Educational, Scientific, and Cultural Organization (ref. SC/ECO/PD/565/ 19.1 1996), Red Latinoamericana de Botánica (95–SP3), and Universidad de Chile (PG–074–96) to L. F. P. We thank C. Mayto, J. Balderrama, and E. Rapu for field assistance. C. Pino wrote the program used for counts of number of alleles in common. D. Sepúlveda, E. Rivera, and numerous people at the Universidad de Chile helped prepare enzyme extracts. D. Sepúlveda and L. Eaton ran the electrophoresis. L. Eguiarte and L. Eaton helped interpret PRX. L. Cardemil and M. Lamborot helped with equipment and suggestions. L. Cardemil’s staff helped with laboratory work. Many people, including park rangers and personnel of the Biosphere Reserve Estación Biológica Beni, biologists, and especially local people helped solve logistical problems. People at the Instituto de Ecología and the Bolivian Government authorities helped to accelerate permits to export plant material to Chile. J. Armesto, R. Bustamante, L. Eaton, L. Eguiarte, K. Holsinger, F. Jaksic, E. Main, I. Serey, E. Vergara, S. Walker, and an anonymous reviewer made useful suggestions to improve this manuscript. The doctoral studies of L. F. P. at the Universidad de Chile were supported by Red Latinoamericana de Botánica (93–D1). Finally, L. F. P. thanks his wife, A. Roldán, for help throughout his dissertation work.


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Literature Cited Allendorf, F. W. 1983. Isolation, gene flow, and genetic differentiation among populations. Pages 51–65 in C. M. Schonewald-Cox, S. M. Chambers, B. MacBryde, and L. Thomas, editors. Genetics and conservation. Cummings Publishing, Menlo Park, California. Alvarez-Buylla, E. R., R. García-Barrios, C. Lara-Moreno, and M. MartínezRamos. 1996. Demographic and genetic models in conservation biology: applications and perspectives for tropical rain forest tree species. Annual Review of Ecology and Systematics 27:387–421. Antonovics, J. 1968. Evolution in closely adjacent plant populations. VI. Manifold effects of gene flow. Heredity 23:507–524. Bawa, K. S. 1990. Plant-pollinator interactions in tropical rain forests. Annual Review of Ecology and Systematics 21:399–422. Candolle, A. P. 1993. Mimosoideae. Pages 420–456 in T. J. Killeen, E. García, and S. G. Beck, editors. Guía de árboles de Bolivia. Herbario Nacional de Bolivia, Missouri Botanical Garden, La Paz, Bolivia. Chambers, S. M. 1983. Genetic principles for managers. Pages 15–46 in C. M. Schonewald-Cox, S. M. Chambers, B. MacBryde, and L. Thomas, editors. Genetics and conservation. Cummings Publishing, Menlo Park, California. Chapman, A. C., and L. J. Chapman. 1995. Survival without dispersers: seedling recruitment under parents. Conservation Biology 9:675–678. Chapman, C. A., and D. A. Onderdonk. 1998. Forests without primates: primate/plant codependency. American Journal of Primatology 45:127–141. Coley, P. D., and J. A. Barone. 1996. Herbivory and plant defenses in tropical forests. Annual Review of Ecology and Systematics 27: 305–335. Conkle, M. T., P. D. Hodgskiss, L. B. Nunnally, and S. C. Hunter. 1982. Starch gel electrophoresis of conifer seeds: a laboratory manual. General technical report PSW-64. Pacific Southwest Forest and Range Experiment Station, U.S. Forest Service, Berkeley, California. Dirzo, R., and C. A. Domínguez. 1986. Seed shadows, seed predation and the advantages of dispersal. Pages 237–249 in A. Estrada and T. H. Fleming, editors. Frugivores and seed dispersal. Dr. W. Junk Publishers, Dordrecht, The Netherlands. Eguiarte, L. E., N. Perez-Nasser, and D. Piñero. 1992. Genetic structure, outcrossing rate and heterosis in Astrocaryum mexicanum (tropical palm): implications for evolution and conservation. Heredity 69:217–228. Ellstrand, N. C., and D. R. Elam. 1993. Population genetic consequences of small population size: implications for plant conservation. Annual Review of Ecology and Systematics 24:217–242. Foster-Huenneke, L. 1991. Ecological implications of genetic variation in plant populations. Pages 31–44 in D. A. Falk and K. E. Holsinger, editors. Genetics and conservation of rare plants. Oxford University Press, New York. García, J. E., and T. Tarifa. 1991. Estudio de la comunidad de primates en la Reserva de la Biosfera “Estación Biológica Beni,” Bolivia. Ecología en Bolivia 17:1–14. Gibson, J. P., and N. T. Wheelwright. 1995. Genetic structure in a population of a tropical tree Ocotea tenera (Lauraceae): influence of avian seed dispersal. Oecologia 103:49–54. Godt, M. J. W., and J. L. Hamrick. 1993. Genetic diversity and population structure in Tradescantia hirsuticaulis (Commelinaceae). American Journal of Botany 80:959–966. Hamrick, J. L. 1982. Plant population genetics and evolution. American Journal of Botany 69:1685–1693. Hamrick, J. L., and M. D. Loveless. 1986. The influence of seed dispersal mechanisms on the genetic structure of plant populations. Pages 211–223 in A. Estrada and T. H. Fleming, editors. Frugivores and seed dispersal. Dr. W. Junk Publishers, Dordrecht, The Netherlands. Hamrick, J. L., and D. A. Murawski. 1990. The breeding structure of tropical tree populations. Plant Species Biology 5:157–165.

Pacheco & Simonetti

Hamrick, J. L., and J. D. Nason. 1996. Consequences of dispersal in plants. Pages 203–236 in O. E. Rhodes Jr., R. K. Chesser, and M. H. Smith, editors. Population dynamics in ecological space and time. The University of Chicago Press, Chicago. Hamrick, J. L., M. J. W. Godt, D. A. Murawski, and M. D. Loveless. 1991. Correlation between species traits and allozyme diversity: implications for conservation biology. Pages 75–86 in D. A. Falk and K. E. Holsinger, editors. Genetics and conservation of rare plants. Oxford University Press, New York. Hamrick, J. L., D. A. Murawski, and J. D. Nason. 1993. The influence of seed dispersal mechanisms on the genetic structure of tropical tree populations. Vegetatio 107/108:281–297. Hartl, D. L. 1990. A primer of population genetics. 2nd edition. Sinauer Associates, Sunderland, Massachusetts. Koptur, S. 1983. Inga (Guaba, Guajinil, Caite, Paterno). Pages 259–261 in D. H. Janzen, editor. Costa Rican natural history. The University of Chicago Press, Chicago. Koptur, S. 1984. Outcrossing and pollinator limitation of fruit set: breeding systems of Neotropical Inga trees (Fabaceae: Mimosoideae). Evolution 38:1130–1143. Lande, R. 1988. Genetics and demography in biological conservation. Science 241:1455–1460. Linhart, Y. B., and M. C. Grant 1996. Evolutionary significance of local genetic differentiation in plants. Annual Review of Ecology and Systematics 27:237–277. Liu, E. H., and M. J. W. Godt. 1983. The differentiation of populations over short distances. Pages 78–95 in C. M. Schonewald-Cox, S. M. Chambers, B. MacBryde, and L. Thomas, editors. Genetics and conservation. Cummings Publishing, Menlo Park, California. Loveless, M. D. 1992. Isozyme variation in tropical trees: patterns of genetic organization. New Forests 6:67–94. Loveless, M. D., and J. L. Hamrick. 1984. Ecological determinants of genetic structure in plant populations. Annual Review of Ecology and Systematics 15:65–95. Miranda, C., M. O. Ribera, J. Sarmiento, E. Salinas, and C. Navia. 1991. Plan de manejo de la Reserva de la Biósfera Estación Biológica del Beni. Academia Nacional de Ciencias Bolivia, Estación Biológica del Beni, Liga de Defensa del Medio Ambiente, PL-480, La Paz, Bolivia. Murphy, R. W., J. W. Sites Jr., D. G. Buth, and C. H. Haufler. 1990. Isozyme electrophoresis. Pages 45–126 in D. M. Hillis and C. Moritz, editors. Molecular systematics. Sinauer Associates, Sunderland, Massachusetts. Nason, J. D., P. R. Aldrich, and J. L. Hamrick. 1997. Dispersal and the dynamics of genetic structure in fragmented tropical tree populations. Pages 304–320 in W. F. Laurance and E. O. Bierregaard Jr., editors. Tropical forest remnants: ecology, management, and conservation of fragmented communities. University of Chicago Press, Chicago. Pacheco, L. F., and J. A. Simonetti. 1998. Consecuencias demográficas para Inga ingoides (Mimosoideae) por la pérdida de Ateles paniscus (Cebidae), uno de sus dispersores de semillas. Ecología en Bolivia 31:67–90. Peres, C. A. 1990. Effects of hunting on western Amazonian primate communities. Biological Conservation 54:47–59. Raymond, M., and F. Rousset. 1995a. An exact test for population differentiation. Evolution 49:1280–1283.


1775

Raymond, M., and F. Rousset. 1995b. GENEPOP (version 1.2): a population genetics software for exact test and ecumenicism. Journal of Heredity 86:248–249. Redford, K. H. 1992. The empty forest. BioScience 42:412–422. Redford, K. H., and J. G. Robinson. 1987. The game of choice: patterns of Indian and colonist hunting in the Neotropics. American Anthropologist 89:650–667. Robinson, J. G., and R. E. Bodmer. 1999. Towards wildlife management in tropical forests. The Journal of Wildlife Management 63:1–13. Roldán, A. I. 1997. El síndrome del bosque vacío ¿Es un fenómeno recurrente en los bosques neotropicales? M.S. thesis. Universidad de Chile, Santiago. Roldán, A. I., M. Moraes, S. Beck, I. Hinojosa, J. Comiskey, and F. Dallmeier. 1999. Lista preliminar de plantas vasculares de la Reserva de la biosfera Estación Biológica del Beni, Bolivia. Documentos, Serie Botánica 4. Ecología en Bolivia: Documentos. Serie Botánica 4:1–24. Roldán, A. I., L. F. Pacheco, and J. A. Simonetti. 2000. Los bosques vacíos de la Reserva de la Biosfera Estación Biológica del Beni, Bolivia. Pages 263–274 in O. Herrera-MacBryde, F. Dallmeier, B. MacBryde, J. A. Comiskey, and C. Miranda, editors. Biodiversidad, conservación y manejo en la región de la Reserva de la Biosfera Estación Biológica del Beni, Bolivia. SI/Monitoring and Assessment of Biodiversity Program Series No. 4, Smithsonian Institution, Washington, D.C. Schnabel, A., and J. L. Hamrick. 1990. Organization of genetic diversity within and among populations of Gleditsia triacanthos (Leguminosae). American Journal of Botany 77:1060–1069. Slatkin, M. 1987. Gene flow and the geographic structure of natural populations. Science 236:787–792. Slatkin, M. 1989. Population structure and evolutionary progress. Genome 31:196–202. Slatkin, M. 1993. Isolation by distance in equilibrium and non-equilibrium populations. Evolution 47:264–279. Surles, S. E., J. Arnold, A. Schnabel, J. L. Hamrick, and B. C. Bongarten. 1990. Genetic relatedness in open-pollinated families of two leguminous tree species, Robinia pseudoacacia L. and Gleditsia triacanthos L. Theoretical and Applied Genetics 80:49–56. Symington, M. M. 1988. Demography, ranging patterns, and activity budgets of black spider monkeys (Ateles paniscus chamek) in the Manu National Park. American Journal of Primatology 15:45–67. van Roosmalen, M. G. M. 1985a. Fruits of the Guianan flora. Institute of Systematic Botany, Utrech University, Utrech, The Netherlands. van Roosmalen. M. G. M. 1985b. Habitat preferences, diet, feeding behavior and social organization of the black spider monkey, Ateles paniscus paniscus in Surinam. Acta Amazonica 15(3–4, supplement):1–238. Weir, B. S., and C. C. Cockerham. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370. Wendel, J. F., and N. F. Weeden. 1989. Visualization and interpretation. Pages 5–45 in D. E. Soltis and P. S. Soltis, editors. Isozymes in plant biology: advances in plant sciences series. Volume 4. Dioscorides Press, Portland, Oregon. Wright, S. 1982. The shifting balance theory and macroevolution. Annual Review of Genetics 16:1–19. Zar, J. H. 1984. Biostatistical Analysis. Prentice-Hall, Englewood, Cliffs, New Jersey.
