All nine microsatellite loci showed high polymorphism levels between categories adults and sub-adults , in both populations. The sub-adult individuals of the fragmented area had a higher value 0. Although the effects were small, a persistent fragmentation process can increase inbreeding and facilitate genetic drift, leading T. Epub March Key words: genetic variability; cacauhy; inbreeding; Amazon; anthropic influence; microsatellite.
Fueron muestreadas las hojas de 75 individuos de T. The Amazon is an extraordinary supplier of natural resources to the Brazilian and world populations. The sustainable use of these resources and the lack of biological knowledge about most of the Amazonian flora species are challenges for future generations Silva et al.
Theobroma speciosum Willd. This species, although little known, is important because it represents a possible source of resistance among most of the economically relevant species belonging to genus Theobroma Silva et al.
Native Theobroma species, such as cacauhy, are losing their habitat due to the intense forest fragmentation in the Amazon, and thus preserving the genetic diversity has been the main goal of most conservation programs focused on preserving the gene of interest and on allowing species variability maintenance at genetic level Bekessy et al.
Forest fragmentation decreases the number of individuals in a given population and, consequently, favors genetic variation losses. Genetic drift is the measured gene frequency of these individuals. In the short term, apart from the gene frequency of the original population, including the allele losses, genetic drift may occur in this small population.
Studying the genetic variation of a given species in a natural population involves two issues: quantifying the variability levels within populations, and characterizing the genetic structure level of these populations Hamrick, According to Hardy-Weinberg, and to the fixation index f , the intrapopulation genetic variability has been quantified through the number of alleles per locus A , the percentage of polymorphic loci P and the observed Ho and expected He heterozygosity Ho Hamrick, The second issue concerns the way genetic variability splits between, and within, populations; in other words, it concerns the genetic structure level of the population.
The genetic structure refers to the heterogeneous distribution non-random of alleles and genotypes in space and time. Such distribution results from the action of evolutionary forces such as mutation, migration, selection and genetic drift within each species and population Hamrick, The lack of information concerning the genetic consequences of forest fragmentation in natural Theobroma speciosum populations led to the aim of the present study, which was to assess whether fragmentation and habitat reduction affect the genetic structure and cause genetic diversity losses in natural T.
The ombrophilous forest is the prevailing vegetation type in the sites. Summers are sunny and winters are clear and dry. The almonds of its berry-type fruits present yellowish color a tripe stage and can be used in soft drinks and chocolate production Figure 1 A. The plant can be successfully used for landscaping due to its exuberant inflorescence along the stem Figure 1 B, Figure 1 C and Figure 1 D Lorenzi, Figure 1 Morphologic aspects of Theobroma speciosum.
A Tree with fruits. B Inflorescence, open flowers. C Inflorescence, flower buds. D Tree with fruits and inflorescence. One natural T. One hundred individuals were sampled in Juruena National Park.
The total genomic DNA was extracted through the cetyltrimethylammonium bromide method by Doyle and Doyle Primer selection and amplification through PCR: A total of 23 microsatellite loci SSR , previously isolated and characterized according to Lanaud et al.
Nine of the twenty-three tested loci were selected for species genetic diversity analysis. The amplification was conducted according to the protocol by Lanaud et al. The genetic diversity of adult and sub-adult individuals was estimated based on the total number of alleles k , on the highest allelic frequency Fa , on the observed H o and expected He heterozygosity at Hardy-Weinberg equilibrium in each locus and across all loci , and on the polymorphism information content PIC , in order to verify the quality of the used loci.
The analysis of variance was conducted in the Genes software Cruz, The inbreeding level between the sampled individuals was estimated based on the fixation index F , according to the method by Weir and Cockerham All the analyses were run in the Power Marker software, version 3. The total genetic diversity was analyzed in the GenAIEx 6. The genetic structure between populations category was also quantified in the PopGene1.
Intrapopulation genetic variability: All nine microsatellite loci have shown high polymorphism levels between categories in the two populations no locus deviated from the Hardy-Weinberg equilibrium proportions. Forty-three individuals per category were analyzed in each population, on average, revealing the total of alleles in the samples. The estimated mean genetic diversity parameter values were very high and homogeneous between categories Table 1.
The number of alleles per locus k ranged from 7. Adults and sub-adults in the JNP population presented the same total number of alleles 78 ; however, adults had more alleles 72 than sub-adults 71 in the population from the urban parks, the adults had 8 unique alleles and the sub-adults, 7.
Although there were no significant differences between values, the contrasting heterozygosity Ho between the two populations evidenced higher heterozygosity Ho in adults and sub-adults from the JNP, and lower heterozygosity Ho in both categories from the urban parks, fact that reveals the inbreeding process in fragmented populations Table 1.
The sub-adult individuals in the fragmented population UF have shown higher F value 0. All loci had high polymorphic information content PIC , the average per category ranged from 0. The allelic frequency analysis has shown that all populations presented unique alleles in the two categories of the herein studied plants. The two groups have shown 09 and 08 unique alleles in the adult and sub-adult individuals divided in the two populations, respectively.
Since the sub-adult individuals in JPN have presented the lowest mean allelic frequency value 0. The sub-adults from the parks have shown the highest mean allele frequency 0. The probabilities were calculated using 1 random permutations.
Table 3 shows that the categories within each T. It is worth highlighting that the adults in the sampled populations are genetically closer to each other than the sub-adults. All loci in the T. According to Botstein , markers presenting PIC above 0. The number of unique alleles in adult individuals suggests the occurrence of genetic drift, i.
Not all individuals are able to have offspring; however, the number of unique alleles in sub-adult individuals suggests lack of gene flow or the presence of parental in the population Carvalho et al. Thus, it appears that the significant changes in the heterozygosity levels of some generations are only possible through drastic effective size reduction; otherwise, a little change can be observed. So far, as the fragmentation in urban park areas is recent less than 40 years , it can be stated that the genetic diversity levels were not affected by the fragmentation process when they were compared to the genetic diversity level shown by plants from the Juruena National Park, which is ruled and supervised by ICMBio.
They found that the remaining populations smaller than 96 trees have shown no signs of genetic variation reduction; thus, suggesting that the effect of genetic drift in a period of years generations is small after forest fragmentation.
Similar results were found in the present study, fact that corroborates the assumption that the intense fragmentation process in the UF did not reduce the allelic frequency in T. The presence of T. The mean fixation index F between loci in the fragmented populations was slightly higher than that of the population in the JNP.
It suggests Hardy-Weinberg equilibrium proportion deviations due to the excess of homozygotes, which was probably caused by inbreeding.
The HWE deviations imply the reproductive division of the population in groups that have a certain degree of relation. The division is possibly associated with family structures, within the population or with preferential mating, as it was observed in the studied populations. Another explanation for the high inbreeding values lies on the presence of null alleles, because it increases the number of homozygous individuals, since just one of the alleles amplifies itself in cases of null allele in heterozygous plant Nybom, Sebbenn et al.
Drosophila sp. The AMOVA results suggest that the genetic differentiation is greater in the intrapopulation component than in the interpopulation one. The values are consistent with those found in other tropical allogamous species. Silva et al. Rossi et al. However, Giustina et al. The genetic distance difference between sub-adults and between adults may result from the recent fragmentation of the study site.
Therefore, the cover-habitat reduction process faced by the forest in the herein studied site has taken place in less than 40 years ago; thus, it appears that fragmentation started to get more intense in the generation sampled in the sub-adult category, since the sampled adults were located in the pre-fragmentation habitat. The genetic information of the nine microsatellite loci have shown that the fragmentation process has so far caused little changes in the diversity levels and in the genetic structure of T.
However, the reduced number of individuals able to reproduce in the population, has resulted in possible mating changes, fact that has led to increased inbreeding levels, mainly in the sub-adults of fragmented populations.
Although the forest fragmentation effects were small, the fragmentation process persistence may further increase the inbreeding levels and facilitate the genetic drift action. These effects may lead the species to inbreeding depression, diversity losses, as well as to changes in the genetic structure of the populations in the course of several generations. Therefore, researches and actions focused on preserving these sites are necessary to avoid genetic diversity losses to keep on happening.
Aldrich, R. Microsatellite analysis of demographic genetic structure in fragmented populations of the tropical tree Symphonia globulifera.
Many plant species rely on animals for dispersal Herrera, ; Ollerton et al. For example, dispersal limitation associated with habitat loss and fragmentation may drive a decline in genetic diversity, an increase in inbreeding and an increase in genetic differentiation among isolated populations Young et al. In contrast, the mating system and fine-scale spatial genetic structure SGS of plant populations may respond immediately or within a single generation Lowe et al.
It is becoming increasingly clear that a suite of changes may be expected post fragmentation and that different genetic characteristics vary in their degree of sensitivity to change Varvio et al. For this reason, there is a need for more empirical data on how different genetic attributes respond to habitat loss and fragmentation to better understand the contemporary and future status of fragmented populations.
For plant species that experience limited gene flow and reduced population sizes following habitat loss and fragmentation, immediate genetic consequences may include the loss of rare alleles, increased inbreeding and increased fine-scale SGS Young et al. These consequences arise from changes in patterns of gene dispersal and the demographic and spatial structure of recruits. The loss of rare alleles and a decline in allelic richness are likely to occur from the immediate loss of individuals following habitat destruction Young et al.
Reduced pollen diversity and increased inbreeding through selfing or mating with close relatives may result from changes in the behavior and distribution of pollinators in fragmented landscapes, contributing to short-term alteration of the realized mating systems of plants Breed et al.
The fine-scale SGS of recruits may also respond in the short-term Wang et al. In contrast, changes to population-level genetic diversity for example, expected heterozygosity and genetic differentiation among subpopulations may require several generations to occur Lowe et al. Theoretical models have shown that the loss of genetic diversity may proceed an order of magnitude more slowly than population differentiation Varvio et al.
Empirical studies have found that populations generally do not show declines in genetic diversity if few generations have passed, except in the most heavily fragmented landscapes Lowe et al.
Extensive gene dispersal via pollen dispersal, seed dispersal or both may prevent genetic drift from eroding genetic diversity and increasing genetic differentiation among fragmented populations Young et al.
Effective dispersal may explain the resilience of certain plant species to the negative genetic consequences of habitat loss and fragmentation Dick, ; White et al. For some species, gene flow may be enhanced in fragmented landscapes as dispersers are forced to move longer distances between plants or habitat patches Dick, ; White et al.
Less is known about the scale and diversity of seed-mediated gene flow in fragmented landscapes Sork and Smouse, ; Hamrick, , though seed movement appears to be sufficient for exchanging individuals between habitat patches in disturbed landscapes in at least some cases Sezen et al.
We examined how habitat loss and fragmentation in northwest Ecuador impacts populations of Oenocarpus bataua , a large-seeded, outcrossing, animal-dispersed palm tree Henderson et al. In doing so, our goal was to advance our understanding of how genetic characteristics of plant populations respond to recent habitat loss and fragmentation.
Our study design was to compare genetic characteristics of O. Our working hypothesis was that allelic richness, genetic differentiation and fine-scale SGS would respond most strongly to this relatively recent fragmentation, whereas within-population gene diversity would not be impacted.
We also hypothesized that inbreeding of O. It produces large inflorescences of thousands of small flowers; the most common pollinators are beetles Curculionidae and bees Meliponinae; Nunez-Avellaneda and Rojas-Robles, Ripe fruits are available for 4—8 weeks and present a large seed Toucans Ramphastos spp.
The Central American agouti Dasyprocta punctata , lowland paca Cuniculus paca , terrestrial birds and smaller rodents remove fallen fruits beneath fruiting trees and provide occasional secondary dispersal L Browne and J Karubian, unpublished data.
Insects in the families Scolytidae bark beetles and Blastobasidae moths are the most common seed predators A. Franzke, unpublished data. Habitat structure of BBS and fragments was qualitatively similar, with both presenting low levels of disturbance. We identified fragments located on private land surrounding BBS within a matrix dominated by pasture, where O. The area of each fragment was obtained by walking fragment borders with a hand-held GPS Garmin, Kansas City, MO, USA because adequate remote-sensing imagery is not currently available for the region see below.
Area ha of each location and distance from BBS are provided in Table 1. To evaluate genetic diversity and structure in BBS and fragments, we sampled different components of the O.
Each recruit sampled had a seed attached to its base, placing all recruits within a similar age class that clearly established post fragmentation. The average height of adults in BBS On the basis of nearly a decade of measuring growth rates of O. For beneath recruits, we sampled multiple individuals from groups of recruits located underneath adult trees. Pairwise and nearest-neighbor distances between sampled individuals were also similar, apart from F15 and F16, which had the smallest sampled area Table 1 and lower pairwise and nearest-neighbor distances Supplementary Figure S1 , Supplementary Table S1.
We genotyped a total of O. Leaf tissue from recruits and tissue from the roots, leaves or surface of the trunk from adults were collected between and and stored in dry conditions until DNA extraction. Genotype data were checked for scoring errors and the presence and frequency of null alleles using Micro-Checker v.
We used A r , H o , H s , F is to compare genetic diversity and inbreeding of adults, beneath recruits and dispersed recruits between fragments and BBS. Unless mentioned otherwise, we pooled adults, beneath recruits and dispersed recruits across fragments; all analyses were also re-run with individuals grouped by fragment Supplementary Table S3. To test for differences in genetic diversity and inbreeding of adults, beneath recruits and dispersed recruits between fragments and BBS, we used a randomized blocked analysis of variance with loci as the blocking factor after arcsine square root transforming H o and H s.
We conducted post hoc comparisons using the Tukey Honest Significant Differences test. For each analysis of variance, we confirmed that model assumptions were met for example, normality of residuals, homogeneity of variance.
These analyses were performed in R v. We evaluated inter-population genetic differentiation of O. We also present F st results to allow comparison with past studies, though F st is limited in estimating genetic differentiation with highly polymorphic markers, such as the ones used in this study Jost, We conducted pairwise comparisons of all the locations for adults, beneath recruits and dispersed recruits separately.
A hierarchical analysis of molecular variance was used to analyze the distribution of genetic variation among and within locations BBS and fragments for each of adults, beneath recruits and dispersed recruits in Arlequin Excoffier and Lischer, To assess the strength of intra-population fine-scale SGS, we performed spatial autocorrelation analysis using the kinship coefficient F ij of Loiselle et al.
Because the sampling size and scale varied between locations, the overall number of intervals varied between 5 and 8 Supplementary Table S4. Adults, beneath recruits and dispersed recruits were pooled across fragments, though analyses were also conducted at the individual fragment level Supplementary Table S4.
For analyses pooled across fragments, reference allele frequencies used in the estimation of F ij were calculated separately for each fragment. For the analyses of individuals pooled across fragments vs BBS, we used equivalent distance intervals to aid in direct comparison. The Sp statistic is robust to the choice of distance intervals Vekemans and Hardy, , but to ensure that our results were not dependent on the arbitrary choice of distance intervals, we recalculated Sp for adults, beneath recruits and dispersed recruits pooled across fragments and in BBS with the number of distance intervals ranging from 2 to 16 and found that indeed our estimates of Sp were robust to the number of chosen distance intervals and size of the first distance class Supplementary Table S5.
These results did not differ substantively from b Flog estimates using the full distance range, and thus we only present those results.
To test whether SGS differed between BBS and fragments, we conducted a randomized blocked analysis of variance using loci as the blocking factor on the per-locus estimates of Sp. We found no significant differences in measures of genetic diversity A r , H o , H s between adults, beneath recruits and dispersed recruits in BBS and fragments pooled, Table 2. When analyzed individually, only one fragment F15 showed consistently lower measures of allelic richness A r and within-population gene diversity H s than in BBS for both adults and recruits Supplementary Table S3.
There was no significant difference in the inbreeding coefficient F is between BBS and fragments pooled for either adults or recruits Table 2. We found significant genetic differentiation among locations BBS and individual fragments for adults, beneath recruits and dispersed recruits.
Pairwise D est between just BBS and the six fragments was again highest in dispersed recruits 0. For both dispersed and beneath recruits, differences among populations 4. Significant fine-scale SGS was found for adults, beneath recruits, and dispersed recruits in BBS, and for beneath and dispersed recruits pooled across fragments Table 3.
Adults pooled across fragments did not show significant SGS Table 3. Understanding how habitat loss and fragmentation impact animal-dispersed plants depends in large part on identifying how genetic characteristics of populations will respond to these forms of disturbance. We found no differences in genetic diversity between fragmented and continuous populations, but we did observe stronger fine-scale SGS among recruits in forest fragments compared with their counterparts in continuous forest.
We also found that genetic differentiation among populations was higher for recruits than for adults, both when comparing across all populations and when comparing BBS directly with fragmented populations. There was no difference in inbreeding in BBS and fragments for recruits or adults, congruent with our prediction on the basis of similar findings from an earlier study Ottewell et al.
These results suggest that, in the short term, genetic diversity can be maintained in fragmented landscapes among early generations of this long-lived palm species, despite increasing levels of within- and between-population genetic structure among recruits. However, we do note that the evidence of increasing genetic differentiation and structure detected may be early warning signals for an anthropogenic impact on the evolutionary trajectory for O.
Genetic diversity of recruits in fragmented and continuous forest was equivalent, congruent with the expectation that genetic diversity will not respond immediately to habitat loss and fragmentation, unless remnant populations are very small and isolated Young et al.
Surprisingly, we found comparable levels of allelic richness, despite it being a particularly sensitive parameter to habitat loss and fragmentation Lowe et al.
One possible explanation is that the genetic bottleneck following deforestation was not severe enough to lower the effective size of remnant populations to a point where loss of genetic diversity would be pronounced Young et al.
This could lead to high standing genetic diversity in adults left after fragmentation, providing a buffer against genetic erosion Hamrick, Only adults and dispersed recruits in the smallest fragment in this study F15, 2. Alternatively, a loss of genetic diversity could be mitigated if between-fragment gene flow via pollen or seeds or both is maintained or even enhanced following fragmentation Sork et al.
However, if gene flow between fragments were maintained or increased, one would also predict that genetic differentiation would remain stable or possibly decrease Hamrick, We detected an increase in genetic differentiation among recruits post fragmentation, suggesting altered patterns of gene flow, but it is difficult to reach any firm conclusions about whether gene flow is increasing or decreasing within the limitations of this current study.
Alternatively, if gene flow across the landscape were limited, we would also expect to see an increase in differentiation among populations, but also a corresponding loss of genetic diversity Young et al. Genetic differentiation might increase without a corresponding loss to diversity if parental densities were reduced to a level that would cause local differentiation in allele frequencies through a random sampling effect without an overall loss of genetic diversity.
The fact that the density of adults for most fragments sampled in this study were higher than in BBS makes this unlikely in this system Supplementary Table S8. A direct analysis of gene flow for example, parentage analysis that quantifies the number of migration events among fragments, along with a better understanding of natural barriers to gene flow in continuous landscapes would be useful to identify the drivers of genetic differentiation in this system.
It is also worth noting that the lack of a detectable response of genetic diversity to fragmentation may be owing to the fact that not enough generations have passed for an effect to be noticeable Kramer et al.
We estimate that the 30—40 years since fragmentation in this landscape corresponds to at the most two generations of O. In a meta-analysis of genetic consequences of fragmentation on plant populations, Aguilar et al. Patterns of fine-scale SGS arise from a complex interaction of factors, including but not limited to the magnitude of pollen and seed dispersal, the pollen and seed pool structure, and population density Vekemans and Hardy, Fine-scale SGS also commonly changes throughout the life cycle of plants, generally strongest at early-life stages and declining over time Hamrick et al.
We recovered a similar pattern in which young recruits had stronger fine-scale SGS than adults in all comparisons, but more interestingly, recruits in fragments had significantly stronger fine-scale SGS than did their counterparts in continuous forest. Lower plant densities can also explain stronger fine-scale SGS Hamrick et al. Interestingly, however, the density of both beneath and dispersed recruits was generally lower in fragments than in BBS Supplementary Table S8 , which may provide a partial explanation of the increase in fine-scale SGS in fragments the mechanisms behind the reduced density of recruits in fragments relative to BBS remain unclear.
The difference in SGS between fragments and BBS was greater for dispersed recruits than for beneath recruits Figure 2 , suggesting that pollen- and seed-mediated gene flow are differentially affected by habitat loss and fragmentation in this system.
If pollen dispersal were impacted by fragmentation, we would expect to see a strong effect on SGS in beneath seedlings in fragments compared with BBS, assuming that beneath recruits in both fragments and BBS experience very limited seed dispersal that is, falling from the infructescence to beneath the adult canopy , and the resulting genetic structure contributed by this limited level of seed dispersal would be similar in both forest types.
Alternatively, if seed dispersal were strongly impacted by fragmentation, we would expect to see a strong difference between fragments and continuous forest in SGS for dispersed recruits, but not necessarily for beneath recruits, because the SGS of dispersed recruits is likely strongly influenced by seed-dispersal processes Wang et al.
A change in the SGS of recruits could arise from a combination of a restriction of dispersal distance of either pollen or seed Vekemans and Hardy, or a change to the pollen and seed pool structure that would result in higher levels of relatedness among dispersed individuals or both. The larger differences in SGS in dispersed recruits between BBS and fragments suggest greater impacts of habitat loss and fragmentation on seed-dispersal processes than pollen dispersal.
However, we suggest caution when interpreting these results as we provide only indirect evidence of this effect, the mechanisms at play remain unclear and we cannot rule out other potential contributing factors like differences in the densities of recruits.
Spatial autocorrelation analysis of Oenocarpus bataua for a adults, b beneath recruits and c dispersed recruits in Bilsa Biological Station BBS, solid gray line with circle and fragments dashed black line with triangle. Relationship coefficient F ij plotted against distance. Lack of replication is a pervasive problem in fragmentation studies Lowe et al. In this study, we compared six fragments to one continuous forest, without replication among continuous forest sites or among landscapes, and careful consideration must be made before extrapolating the conclusions of this study beyond the sampled populations.
It is also worth noting that, due to a lack of cloud-free remote-sensing images, we were not able to obtain a land-cover map of the study area, and thus were unable to characterize the matrix between fragments and map the boundaries of all fragments in the region.
Consequently, we were not able assess how fragment isolation and matrix relate to the results of this study, though they are known to be important in other systems Prugh et al. Finally, we were not able to distinguish between the separate processes of habitat loss and habitat fragmentation Fahrig, , although, in our study area, these phenomena typically occur in tandem and thus it is their combined effects that pose a potential threat to the evolutionary potential of O.
In summary, we provide evidence of differential short-term responses of genetic characteristics to a recent fragmentation event: fine-scale SGS and genetic differentiation responded strongly, whereas genetic diversity and inbreeding did not show a strong response. Fine-scale SGS, in particular, appears to be a sensitive and useful tool for detecting genetic changes following habitat loss and fragmentation, though additional study is needed to discern causative mechanisms.
Many tropical tree species rely on animals for dispersal of pollen and seed Herrera, ; Ollerton et al. Directly measuring the relative importance of gene flow via pollen vs seed dispersal, identifying how landscape characteristics and matrix quality influence gene flow and conducting comparative longitudinal analyses are priorities for future work on this system.
Genetic consequences of habitat fragmentation in plant populations: susceptible signals in plant traits and methodological approaches. Mol Ecol 17 : — Article Google Scholar. Pollen diversity matters: revealing the neglected effect of pollen diversity on fitness in fragmented landscapes.
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