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Allele frequencies for each locus. Alleles present in very low numbers have the frequency presented above the allele if the bar too small to be easily visualized. All potential allele sizes within the captured range (assuming normal dinucleotide repeat units) are included in the x-axis; blanks indicate that this allele size was not captured. A dot below the allele indicates an unusual size (1 bp difference from nearest allele). ‘a’ indicates a private allele in the adult age class, ‘s’ indicates a private allele in the seedling cohort. doi:10.1371/journal.pone.0082632.g002
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Over the last 150 years, Singapore's primary forest has been reduced to less than 0.2% of its previous area, resulting in extinctions of native flora and fauna. Remaining species may be threatened by genetic erosion and inbreeding. We surveyed >95% of the remaining primary forest in Singapore and used eight highly polymorphic microsatellite loci to...
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... ( Table 2). The large number of rare alleles and the few allele size gaps in most loci support this conclusion (Figure 2). Rare alleles ( , 1%) were most common in tails of the size distribution for most loci (Figure 2) and comprised 30.1% of the total data set (Figure 3). Allele accumulation curves (Figure 4) indicated that we likely captured the majority of the alleles present at these loci in K. malaccensis within Singapore. All three cohorts had spatial genetic autocorrelograms that deviated significantly from the null hypothesis, i.e. all had significant genetic structure (p , 0.001 for all) (Figure 5). The SGS of the adult cohort was significantly different from the seedling and sapling cohorts (p , 0.001), although the seedling and sapling cohorts were not significantly different (p = 0.30). In addition, the overall mean relatedness (r) at the 0–10 m distance class for the adult cohort was approximately half that of the recruit cohorts; this difference was significant between adults and the seedling and sapling cohorts (p = 0.021 and 0.029, respectively) although not between the seedlings and saplings (p = 0.36) (Table 2). S p was lowest in the adult cohort (0.0044), and increased with younger cohorts (saplings = 0.0131, seedlings = 0.0287) (Table 2). The non-overlapping confidence intervals indicate significant differences in S p between each of the three age classes. Gene flow and intergenerational studies using exhaustively surveyed landscapes are crucially lacking in the tropical tree literature [50]. In the few recent studies using comprehensive surveys (i.e. . 95% of the population likely captured) for formerly widespread tree species, significant reductions in genetic diversity were most often detected in the recruit cohort [22], [23], [24]. Thus, our results stand in contrast to theoretical predictions as well as many experimental studies of the genetic consequences of past deforestation (reviewed in [22]). This study detected neither a genetic bottleneck nor a reduction in effective population size, as well as high genetic diversity and a large proportion of rare alleles in all age classes. Genetic studies of K. malaccensis in Malaysia [33], [34], [35] provide some reference points (Table 2) and indicate that genetic diversity of K. malaccensis in Singapore is generally on par with populations studied in more intact Malaysian forests. While these unexpected results may not be representative of the majority of primary forest species (and demographic factors are still a cause for concern) they demonstrate lower than expected genetic vulnerability for a locally threatened, primary forest tree species in a highly urbanized landscape. The initial decline in genetic diversity during habitat loss is due to demographic factors, i.e. the removal of individuals. Subse- quently, genetic drift and reduced genetic diversity generally results from only a subset of individuals contributing gametes to the next generation, e.g. [25], [51]. However the genetic diversity detected for K. malaccensis in Singapore would be considered high even for common, widespread species in pristine habitats [21] and there were no significant differences in genetic indices between age classes. We captured a large fraction of all alleles based on the accumulation curves (Figure 4): alleles with a frequency of less than 1% constituted 30.1% of the data set, and 84.6% of the total number of alleles had a frequency of less than 10% (Figure 3). In addition, there were few gaps in the size distribution (Figure 2), which is contrary to theoretical expectations as well as experimental studies that have documented a loss of alleles during severe population decline [52], [53]. The majority ( , 99%) of alleles present in the adult population were also present in the recruit cohort, in addition to twelve private alleles in the seedling cohort (Figure 2). Given that all private alleles were PCRed at least twice, genotyping errors were unlikely to have contributed substantially to these results. Explanations for these rare, putatively private alleles in the seedlings could include the following: 1. Unsampled adult trees containing these rare alleles within the existing primary forest fragments; 2. Gene flow from isolated trees containing these rare alleles located within smaller forest fragments in the intervening landscape or remnant trees in Singapore’s urbanized matrix; 3. Spontaneous mutations, which could account for some of the private alleles given that , 2,000 seedlings were genotyped, and experimental mutation rates for dinucleotide repeats in plant species is estimated to be , 2.4 6 10 2 4 mutations/generation/locus [54]. Low inbreeding and high heterozygosity are positive indicators for short-term population viability; high allelic richness and a large proportion of rare alleles indicate an adequately large effective population size for long-term viability [55]. While only a relatively small number of individuals may be needed in the short term to prevent inbreeding, greater genetic variation is thought to increase the likelihood of survival over much longer time scales [11]. Genetic diversity measured via neutral microsatellite ...
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... ( Table 2). The large number of rare alleles and the few allele size gaps in most loci support this conclusion (Figure 2). Rare alleles ( , 1%) were most common in tails of the size distribution for most loci (Figure 2) and comprised 30.1% of the total data set (Figure 3). Allele accumulation curves (Figure 4) indicated that we likely captured the majority of the alleles present at these loci in K. malaccensis within Singapore. All three cohorts had spatial genetic autocorrelograms that deviated significantly from the null hypothesis, i.e. all had significant genetic structure (p , 0.001 for all) (Figure 5). The SGS of the adult cohort was significantly different from the seedling and sapling cohorts (p , 0.001), although the seedling and sapling cohorts were not significantly different (p = 0.30). In addition, the overall mean relatedness (r) at the 0–10 m distance class for the adult cohort was approximately half that of the recruit cohorts; this difference was significant between adults and the seedling and sapling cohorts (p = 0.021 and 0.029, respectively) although not between the seedlings and saplings (p = 0.36) (Table 2). S p was lowest in the adult cohort (0.0044), and increased with younger cohorts (saplings = 0.0131, seedlings = 0.0287) (Table 2). The non-overlapping confidence intervals indicate significant differences in S p between each of the three age classes. Gene flow and intergenerational studies using exhaustively surveyed landscapes are crucially lacking in the tropical tree literature [50]. In the few recent studies using comprehensive surveys (i.e. . 95% of the population likely captured) for formerly widespread tree species, significant reductions in genetic diversity were most often detected in the recruit cohort [22], [23], [24]. Thus, our results stand in contrast to theoretical predictions as well as many experimental studies of the genetic consequences of past deforestation (reviewed in [22]). This study detected neither a genetic bottleneck nor a reduction in effective population size, as well as high genetic diversity and a large proportion of rare alleles in all age classes. Genetic studies of K. malaccensis in Malaysia [33], [34], [35] provide some reference points (Table 2) and indicate that genetic diversity of K. malaccensis in Singapore is generally on par with populations studied in more intact Malaysian forests. While these unexpected results may not be representative of the majority of primary forest species (and demographic factors are still a cause for concern) they demonstrate lower than expected genetic vulnerability for a locally threatened, primary forest tree species in a highly urbanized landscape. The initial decline in genetic diversity during habitat loss is due to demographic factors, i.e. the removal of individuals. Subse- quently, genetic drift and reduced genetic diversity generally results from only a subset of individuals contributing gametes to the next generation, e.g. [25], [51]. However the genetic diversity detected for K. malaccensis in Singapore would be considered high even for common, widespread species in pristine habitats [21] and there were no significant differences in genetic indices between age classes. We captured a large fraction of all alleles based on the accumulation curves (Figure 4): alleles with a frequency of less than 1% constituted 30.1% of the data set, and 84.6% of the total number of alleles had a frequency of less than 10% (Figure 3). In addition, there were few gaps in the size distribution (Figure 2), which is contrary to theoretical expectations as well as experimental studies that have documented a loss of alleles during severe population decline [52], [53]. The majority ( , 99%) of alleles present in the adult population were also present in the recruit cohort, in addition to twelve private alleles in the seedling cohort (Figure 2). Given that all private alleles were PCRed at least twice, genotyping errors were unlikely to have contributed substantially to these results. Explanations for these rare, putatively private alleles in the seedlings could include the following: 1. Unsampled adult trees containing these rare alleles within the existing primary forest fragments; 2. Gene flow from isolated trees containing these rare alleles located within smaller forest fragments in the intervening landscape or remnant trees in Singapore’s urbanized matrix; 3. Spontaneous mutations, which could account for some of the private alleles given that , 2,000 seedlings were genotyped, and experimental mutation rates for dinucleotide repeats in plant species is estimated to be , 2.4 6 10 2 4 mutations/generation/locus [54]. Low inbreeding and high heterozygosity are positive indicators for short-term population viability; high allelic richness and a large proportion of rare alleles indicate an adequately large effective population size for long-term viability [55]. While only a relatively small number of individuals may be needed in the short term to prevent inbreeding, greater genetic variation is thought to increase the likelihood of survival over much longer time scales [11]. Genetic diversity measured via neutral microsatellite markers is likely correlated to genetic variation at functional genes [56], and adaptation or resilience to novel or changing habitats is most often positively correlated to genetic variation [57], [58]. Thus, management strategies should be implemented that maintain as far as possible the full complement of genetic diversity within Singapore. In the present study, the adult cohort had low SGS intensity (S p = 0.0044) similar to other tropical tree species (see review of S p values for trees in [59]). This corresponds to recent research showing that long-lived tropical rainforest canopy tree species pollinated by the highly mobile, migratory bee species Apis dorsata exhibited less SGS compared to species pollinated by smaller, less mobile pollinators [60]. Thus, a signature of frequent long- distance pollen dispersal may remain in the adult cohort. In contrast, the recruit cohorts had significantly different SGS autocorrelogram structure compared to the adult cohort and significantly higher mean relatedness in the 0–10 m distance class. Moreover, SGS intensity significantly increased as cohorts got younger (Table 2). While the results provide only a one-time snapshot of the spatial genetic structure, they could point to one of two trends. First, the results could indicate that recruit cohorts of K. malaccensis are showing sensitivity to habitat loss (e.g. [26]). If this was the case, S p and mean relatedness in the recruit cohorts would remain comparatively higher than the present adult cohort as they mature. Alternatively, given that the majority of seedlings and saplings die before reaching maturity, the present-day recruit cohorts may converge towards adult SGS intensities as their numbers undergo thinning (e.g. [27]). Long-term population monitoring would be necessary to determine which explanation is most fitting. The overall outcrossing rate (t m ) for the whole dataset as well as the subset was as high as in the Malaysian populations [34]. However, it is possible that our sampling strategy (see Methods) could have led to a slight inflation of outcrossing rate for the Singapore population. In contrast, the biparental inbreeding rate (t m -t s ) was much higher overall in Singapore than in Malaysia (Table 2), indicating that trees with higher relatedness are mating at a greater frequency compared to the Malaysian population. While this interpretation would not be unexpected given our other results, there could be confounding variables that reduce the validity of this comparison. In particular, the spatial distance between the trees in the Malaysia study was not reported (they were ‘‘distant’’ from each other [34]), and hence the difference in values could simply reflect that a larger number of adjacent adult trees were sampled in Singapore, with the likely consequence of detecting matings between more closely related neighbours. This speculation is supported by the results of a random subsample of 9 Singapore adults (analyzed using only 4 loci, one of which was the same between studies), in which the biparental inbreeding estimates were on par with the Malaysian population (Table 2). Regardless of comparisons to Malaysia, the overall data from Singapore showed a high level of outcrossing, but the moderate level of outcrossing occurring between related adults could ultimately increase the level of inbreeding. Once a population reaches a critically low threshold of individuals, demographics can play an equal—if not more important—role in long-term population persistence compared to genetic factors, although the two are often inexorably linked [57], [61]. A previous study estimating a universal minimum ...
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... ( Table 2). The large number of rare alleles and the few allele size gaps in most loci support this conclusion (Figure 2). Rare alleles ( , 1%) were most common in tails of the size distribution for most loci (Figure 2) and comprised 30.1% of the total data set (Figure 3). Allele accumulation curves (Figure 4) indicated that we likely captured the majority of the alleles present at these loci in K. malaccensis within Singapore. All three cohorts had spatial genetic autocorrelograms that deviated significantly from the null hypothesis, i.e. all had significant genetic structure (p , 0.001 for all) (Figure 5). The SGS of the adult cohort was significantly different from the seedling and sapling cohorts (p , 0.001), although the seedling and sapling cohorts were not significantly different (p = 0.30). In addition, the overall mean relatedness (r) at the 0–10 m distance class for the adult cohort was approximately half that of the recruit cohorts; this difference was significant between adults and the seedling and sapling cohorts (p = 0.021 and 0.029, respectively) although not between the seedlings and saplings (p = 0.36) (Table 2). S p was lowest in the adult cohort (0.0044), and increased with younger cohorts (saplings = 0.0131, seedlings = 0.0287) (Table 2). The non-overlapping confidence intervals indicate significant differences in S p between each of the three age classes. Gene flow and intergenerational studies using exhaustively surveyed landscapes are crucially lacking in the tropical tree literature [50]. In the few recent studies using comprehensive surveys (i.e. . 95% of the population likely captured) for formerly widespread tree species, significant reductions in genetic diversity were most often detected in the recruit cohort [22], [23], [24]. Thus, our results stand in contrast to theoretical predictions as well as many experimental studies of the genetic consequences of past deforestation (reviewed in [22]). This study detected neither a genetic bottleneck nor a reduction in effective population size, as well as high genetic diversity and a large proportion of rare alleles in all age classes. Genetic studies of K. malaccensis in Malaysia [33], [34], [35] provide some reference points (Table 2) and indicate that genetic diversity of K. malaccensis in Singapore is generally on par with populations studied in more intact Malaysian forests. While these unexpected results may not be representative of the majority of primary forest species (and demographic factors are still a cause for concern) they demonstrate lower than expected genetic vulnerability for a locally threatened, primary forest tree species in a highly urbanized landscape. The initial decline in genetic diversity during habitat loss is due to demographic factors, i.e. the removal of individuals. Subse- quently, genetic drift and reduced genetic diversity generally results from only a subset of individuals contributing gametes to the next generation, e.g. [25], [51]. However the genetic diversity detected for K. malaccensis in Singapore would be considered high even for common, widespread species in pristine habitats [21] and there were no significant differences in genetic indices between age classes. We captured a large fraction of all alleles based on the accumulation curves (Figure 4): alleles with a frequency of less than 1% constituted 30.1% of the data set, and 84.6% of the total number of alleles had a frequency of less than 10% (Figure 3). In addition, there were few gaps in the size distribution (Figure 2), which is contrary to theoretical expectations as well as experimental studies that have documented a loss of alleles during severe population decline [52], [53]. The majority ( , 99%) of alleles present in the adult population were also present in the recruit cohort, in addition to twelve private alleles in the seedling cohort (Figure 2). Given that all private alleles were PCRed at least twice, genotyping errors were unlikely to have contributed substantially to these results. Explanations for these rare, putatively private alleles in the seedlings could include the following: 1. Unsampled adult trees containing these rare alleles within the existing primary forest fragments; 2. Gene flow from isolated trees containing these rare alleles located within smaller forest fragments in the intervening landscape or remnant trees in Singapore’s urbanized matrix; 3. Spontaneous mutations, which could account for some of the private alleles given that , 2,000 seedlings were genotyped, and experimental mutation rates for dinucleotide repeats in plant species is estimated to be , 2.4 6 10 2 4 mutations/generation/locus [54]. Low inbreeding and high heterozygosity are positive indicators for short-term population viability; high allelic richness and a large proportion of rare alleles indicate an adequately large effective population size for long-term viability [55]. While only a relatively small number of individuals may be needed in the short term to prevent inbreeding, greater genetic variation is thought to increase the likelihood of survival over much longer time scales [11]. Genetic diversity measured via neutral microsatellite markers is likely correlated to genetic variation at functional genes [56], and adaptation or resilience to novel or changing habitats is most often positively correlated to genetic variation [57], [58]. Thus, management strategies should be implemented that maintain as far as possible the full complement of genetic diversity within Singapore. In the present study, the adult cohort had low SGS intensity (S p = 0.0044) similar to other tropical tree species (see review of S p values for trees in [59]). This corresponds to recent research showing that long-lived tropical rainforest canopy tree species pollinated by the highly mobile, migratory bee species Apis dorsata exhibited less SGS compared to species pollinated by smaller, less mobile pollinators [60]. Thus, a signature of frequent long- distance pollen dispersal may remain in the adult cohort. In contrast, the recruit cohorts had significantly different SGS autocorrelogram structure compared to the adult cohort and significantly higher mean relatedness in the 0–10 m distance class. Moreover, SGS intensity significantly increased as cohorts got younger (Table 2). While the results provide only a one-time snapshot of the spatial genetic structure, they could point to one of two trends. First, the results could indicate that recruit cohorts of K. malaccensis are showing sensitivity to habitat loss (e.g. [26]). If this was the case, S p and mean relatedness in the recruit cohorts would remain comparatively higher than the present adult cohort as they mature. Alternatively, given that the majority of seedlings and saplings die before reaching maturity, the present-day recruit cohorts may converge towards adult SGS intensities as their numbers undergo thinning (e.g. [27]). Long-term population monitoring would be necessary to determine which explanation is most fitting. The overall outcrossing rate (t m ) for the whole dataset as well as the subset was as high as in the Malaysian populations [34]. However, it is possible that our sampling strategy (see Methods) could have led to a slight inflation of outcrossing rate for the Singapore population. In contrast, the biparental inbreeding rate (t m -t s ) was much higher overall in Singapore than in Malaysia (Table 2), indicating that trees with higher relatedness are mating at a greater frequency compared to the Malaysian population. While this interpretation would not be unexpected given our other results, there could be confounding variables that reduce the validity of this comparison. In particular, the spatial distance between the trees in the Malaysia study was not reported (they were ‘‘distant’’ from each other [34]), and hence the difference in values could simply reflect that a larger number of adjacent adult trees were sampled in Singapore, with the likely consequence of detecting matings between more closely related neighbours. This speculation is supported by the results of a random subsample of 9 Singapore adults (analyzed using only 4 loci, one of which was the same between studies), in which the biparental inbreeding estimates were on par with the Malaysian population (Table 2). Regardless of comparisons to Malaysia, the overall data from Singapore showed a high level of outcrossing, but the moderate level of outcrossing occurring between related adults could ...
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... ( Table 2). The large number of rare alleles and the few allele size gaps in most loci support this conclusion (Figure 2). Rare alleles ( , 1%) were most common in tails of the size distribution for most loci (Figure 2) and comprised 30.1% of the total data set (Figure 3). Allele accumulation curves (Figure 4) indicated that we likely captured the majority of the alleles present at these loci in K. malaccensis within Singapore. All three cohorts had spatial genetic autocorrelograms that deviated significantly from the null hypothesis, i.e. all had significant genetic structure (p , 0.001 for all) (Figure 5). The SGS of the adult cohort was significantly different from the seedling and sapling cohorts (p , 0.001), although the seedling and sapling cohorts were not significantly different (p = 0.30). In addition, the overall mean relatedness (r) at the 0–10 m distance class for the adult cohort was approximately half that of the recruit cohorts; this difference was significant between adults and the seedling and sapling cohorts (p = 0.021 and 0.029, respectively) although not between the seedlings and saplings (p = 0.36) (Table 2). S p was lowest in the adult cohort (0.0044), and increased with younger cohorts (saplings = 0.0131, seedlings = 0.0287) (Table 2). The non-overlapping confidence intervals indicate significant differences in S p between each of the three age classes. Gene flow and intergenerational studies using exhaustively surveyed landscapes are crucially lacking in the tropical tree literature [50]. In the few recent studies using comprehensive surveys (i.e. . 95% of the population likely captured) for formerly widespread tree species, significant reductions in genetic diversity were most often detected in the recruit cohort [22], [23], [24]. Thus, our results stand in contrast to theoretical predictions as well as many experimental studies of the genetic consequences of past deforestation (reviewed in [22]). This study detected neither a genetic bottleneck nor a reduction in effective population size, as well as high genetic diversity and a large proportion of rare alleles in all age classes. Genetic studies of K. malaccensis in Malaysia [33], [34], [35] provide some reference points (Table 2) and indicate that genetic diversity of K. malaccensis in Singapore is generally on par with populations studied in more intact Malaysian forests. While these unexpected results may not be representative of the majority of primary forest species (and demographic factors are still a cause for concern) they demonstrate lower than expected genetic vulnerability for a locally threatened, primary forest tree species in a highly urbanized landscape. The initial decline in genetic diversity during habitat loss is due to demographic factors, i.e. the removal of individuals. Subse- quently, genetic drift and reduced genetic diversity generally results from only a subset of individuals contributing gametes to the next generation, e.g. [25], [51]. However the genetic diversity detected for K. malaccensis in Singapore would be considered high even for common, widespread species in pristine habitats [21] and there were no significant differences in genetic indices between age classes. We captured a large fraction of all alleles based on the accumulation curves (Figure 4): alleles with a frequency of less than 1% constituted 30.1% of the data set, and 84.6% of the total number of alleles had a frequency of less than 10% (Figure 3). In addition, there were few gaps in the size distribution (Figure 2), which is contrary to theoretical expectations as well as experimental studies that have documented a loss of alleles during severe population decline [52], [53]. The majority ( , 99%) of alleles present in the adult population were also present in the recruit cohort, in addition to twelve private alleles in the seedling cohort (Figure 2). Given that all private alleles were PCRed at least twice, genotyping errors were unlikely to have contributed substantially to these results. Explanations for these rare, putatively private alleles in the seedlings could include the following: 1. Unsampled adult trees containing these rare alleles within the existing primary forest fragments; 2. Gene flow from isolated trees containing these rare alleles located within smaller forest fragments in the intervening landscape or remnant trees in Singapore’s urbanized matrix; 3. Spontaneous mutations, which could account for some of the private alleles given that , 2,000 seedlings were genotyped, and experimental mutation rates for dinucleotide repeats in plant species is estimated to be , 2.4 6 10 2 4 mutations/generation/locus [54]. Low inbreeding and high heterozygosity are positive indicators for short-term population viability; high allelic richness and a large proportion of rare alleles indicate an adequately large effective population size for long-term viability [55]. While only a relatively small number of individuals may be needed in the short term to prevent inbreeding, greater genetic variation is thought to increase the likelihood of ...
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... GeneMapper v. 4.1. One-third of adults and 0.7% of the recruit cohorts were re-extracted, PCRed and genotyped in their entirety, and 100% of adults and 24.9% of the recruit cohorts were re-PCRed and genotyped at 1 to 8 loci to confirm rare alleles as well as to estimate genotyping errors. Genotyping errors were calculated per locus as the percent of non- matching alleles in a second PCR (Table 1). Microchecker v. 2.2.3 [39] was used to detect the presence of null alleles as well as possible technical scoring errors. Given the high levels of null alleles detected in locus Km071, this locus was excluded from the calculations of F IS values (the inbreeding coefficient, used to detect inbreeding), as the conclusions drawn would not be biologically accurate—i.e. a high F IS value at this locus would not reflect inbreeding. To evaluate the adequacy of our field sampling in capturing the full range of allelic diversity in Singapore, we produced curves for each locus depicting the accumulation of alleles as a function of the number of samples analyzed. Allelic richness (R), expected and observed heterozygosity (H e , H o ) and inbreeding co-efficients (F IS ) were calculated with Fstat v. 2.9.3 [40]. H e and R among age classes were compared with an Analysis of Variance at the locus level. For mating system calculations, seedlings or saplings were classified as the progeny of the nearest adult K. malaccensis . Mltr v. 3.4 [41] was used to calculate outcrossing rates (t m ) and biparental inbreeding rates (t m t s ) under default parameters and with the possibility of null alleles; the putative maternal genotype for each set of putative offspring was included. Four chosen loci (Km011, Km127, Km158a, Km180) were analysed for a random subsample of 9 adults in order to compare the mltr results from the Singapore population to the Malaysia population under more analogous parameters. M_P_val v. 2 [42] was used to detect a potential reduction in effective population size in the adult as well as recruit cohorts, as theory predicts that a population reduction of 50% is expected to lead to a 20% loss of discrete allele sizes and would correspond to gaps in the allele size distribution [42]. An M-ratio value , 0.70 generally indicates that a population has undergone a reduction in effective population size, whereas a population with an M-ratio value . 0.80 indicates population stability. The program Bottleneck [43] utilizes the assumption that rare alleles will be lost more quickly than heterozygotes during a bottleneck, and detects a mode-shift under conditions consistent with a recent bottleneck. Depending on the initial population size, the decay in the genetic signature of bottleneck detection under random mating means that bottlenecks can usually be detected no more than 2 to 3 generations prior to the current generation, whereas M_P_val can be used to detect more historical population size reductions. Bottleneck v. 1.2.02 was run for adult, juvenile and seedling populations respectively, using the infinite allele and stepwise mutation models with 10,000 iterations per run. Spatial genetic structure (SGS) for the adults, saplings, and seedlings was analyzed with GenAlex v. 6.5.3 [44] under the following parameters: 999 permutations and 999 bootstraps, user- defined distance classes (5, 10, 20, 40, 80, 160, 320, 640, 1280, 2560, and 5120 m)—except no 5 m distance class was possible for the adult cohort due to too few individuals within this distance class. Significance of autocorrelogram results (testing both the null hypothesis of no structure for each cohort as well as significant differences between cohorts) was determined using the ‘‘Multiple pops’’ option using the above parameters and the adult cohort distance classes (i.e. 0–10 m for the first distance class). The relatedness metric ‘‘r’’ (Queller & Goodnight) [45] was also obtained from GenAlex; full siblings have r-values of 0.50 and half siblings, 0.25. SpaGeDi v. 1.4 [46] was used to determine spatial genetic structure intensity (S p ) using the formula -b/(1-F 1 ), where b is the slope of the regression between pairwise kinship coefficients [47] and the logarithm of spatial distance, and F 1 is the mean pairwise kinship coefficient within the first distance class (see [48]). Identical (adult) distances classes were entered for all three cohorts. Approximate 95% confidence intervals for S p were calculated as 6 2 6 the standard error of b [49]. Calculations were performed with 9999 permutations and jackknifing over loci. Genetic diversity was high and not significantly different between age classes (H e = 0.843–0.854, p = 0.98; R = 16.72– 19.45, p = 0.20) (Table 2). The inbreeding co-efficient values for all three age classes were low (F IS = 0.013–0.076) and not significant (p = 0.18), although there was an upward trend in younger age classes (Table 2). There were twelve private alleles in the seedling cohort and two private alleles in the adult cohort (Figure 2). Over the whole dataset, the multilocus outcrossing rate was high (t m = 0.961, SD 0.015) and the biparental inbreeding rate (t m -t s ) was 0.111 (SD 0.011) (Table 2). We detected neither a genetic bottleneck in any age class (no mode shift) nor a reduction in effective population size (M- ratio = 0.857 and 0.957 for the adult and recruit ...
Citations
... By examining genetic changes over time through temporal sampling (where material from individuals that pre-date the impact of interest should be sampled, as well as populations that established after the impact has had the opportunity to affect diversity; Leigh et al. 2021), researchers may gain valuable insights into the adaptive potential of populations, the impact of environmental factors on genetic diversity, and the efficacy of conservation interventions (Díez-del-Molino et al. 2018). While microsatellite markers have traditionally been used to survey genetic variation across age classes (Kettle et al. 2007;Noreen and Webb 2013), the advent of next-generation sequencing (NGS) data offers several advantages to study the conservation genomics of non-model organisms. NGS methods such as genotyping by sequencing (GBS; Hall et al. 2020) or double-digest restriction-site associated DNA (ddRAD) sequencing (Peterson et al. 2012), can provide useful snapshots of genomic variation across the entire genome, allowing for the assessment of genetic variation across a wide range of loci, and fine-scale resolution of genetic diversity and population structure (Hauser et al. 2011;Niissalo et al. 2018;Yang et al. 2022). ...
... Very few conservation genetics studies using NGS techniques have been carried out to assess Southeast Asian tropical trees. The assessment of genetic diversity and structure is limited to Dipterocarpaceae species in this region (Ng et al. 2019;Ohtani et al. 2021;Ogasahara et al. 2023) and the only other study of intergenerational genetic characteristics in a native Singapore tree species was carried out on Koompasia malaccensis (Noreen and Webb 2013), all based on characterising between eight and around 20 microsatellite markers. The K. malaccensis study concluded that there is high genetic diversity in all age classes, contrary to expectations of fragmentation-induced genetic erosion, although there were some signs of reduced gene flow in younger cohorts. ...
... In the only other study investigating possible genetic erosion of a forest tree species in Singapore, Noreen and Webb (2013) found high genetic diversity in Koompassia malaccensis, with connectivity between BTNR, CCNR and the Singapore Botanic Gardens facilitated by a mobile pollinator, the giant honeybee Apis dorsata (Noreen et al. 2016). However, there were already signs of increased relatedness between individuals at short distances. ...
Comparing the genetic diversity across different generations within tropical tree populations is an understudied topic. To assess the potential genetic consequences and conservation implications of contemporary disturbances, a population genomic study of Palaquium obovatum across age classes was undertaken. Trees and juveniles were sampled from ten different localities (eight in Singapore, two in Peninsular Malaysia) and subjected to double digest restriction-site associated DNA-sequencing (ddRAD-seq) to assess intergenerational genetic differences and investigate population structure in a hexaploid lineage. Genetic erosion, characterised by reduced heterozygosity, was found to have occurred in almost all wild populations over time, the exceptions being in one isolated coastal population and some areas with cultivated occurences. Population structure was highly localised with the number of genetically distinct populations usually following geographically separated districts, which indicates limitations in pollen and seed dispersal between fragments, possibly due to declines in the associated assemblage of dispersers. For this reason, the germplasm for conserving species diversity in degraded habitats and forest fragments should be selected from a wide range of wild populations across the landscape.
... Consequently, high genetic diversity has been reported in many trees, such as Koompassia malaccensis Maingay ex Benth. [9], Cercis canadensis L. [10], and Shorea leprosula Miq. [11]. ...
The diversity of genetic resources is essential to cope with environmental changes. However, despite forests play a crucial role in mitigating changes, genetic knowledge has scarcely been used for forest conservation. In this study, we used nuclear microsatellites to understand the patterns of genetic diversity and population genetic structure in Ocotea rotundata van der Werff (Lauraceae), an endemic Ecuadorian tree, highly affected by habitat changes and fragmentation. Our results show high levels of genetic diversity, except in one population. The level of genetic differentiation between populations was low and genetic clusters showed no apparent spatial pattern. In fact, a high degree of genetic admixture was found between most populations. Migration rates were asymmetric but overall high, except in one population, where outgoing gene dispersal was limited. Nevertheless, allelic fixation values suggested a general deficit in heterozygotes, probably due to an increase in the levels of mating between close relatives. Although long-lived organisms, such as trees, can often accumulate a surprising amount of genetic diversity, the results found here could be an early sign of a decline in the diversity of O. rotundata. These findings provide baseline information on genetic resources to support future restoration programs to mitigate the impacts of changes in O. rotundata populations.
... This hybrid individual was observed to have been pollinated by the giant honey bee (Apis dorsata). These bees are known to be able to transfer pollen over sizeable distances, and have also been inferred to facilitate pollen dispersal of another leguminous tree species, Koompassia malaccensis, between rainforest patches across a distance of more than 2.5 km within the urban landscape of Singapore (Noreen and Webb 2013;Noreen et al. 2016). Given that these bees are likely to be pollinators of other Sindora species, it is therefore possible that this hybrid could have come about by the transfer of pollen between S. coriacea and S. echinocalyx trees at Changi in the past. ...
Sindora × changiensis L.M.Choo, Loo, W.F.Ang & K.Er is a new hybrid from the subfamily Detarioideae in Fabaceae. This is the first reported instance of natural hybridisation in Sindora. Based on population genetics analyses using ddRAD and morphological observations, this taxon represents a fertile hybrid between Sindora coriacea and Sindora echinocalyx. This new hybrid is so far only known to occur naturally from Changi at the northeastern coast of Singapore. It has pods that are sparsely spiny. This is intermediate between the smooth, non-spiny pods of S. coriacea, and the densely spiny pods of S. echinocalyx. The calyx is smooth and unarmed, resembling S. coriacea. Last but not least, the ovary is entirely pubescent, different from S. coriacea and S. echinocalyx. The ovary of S. coriacea has a glabrous patch in the middle , while that of S. echinocalyx has minute spines protruding from the dense pubescence. A taxonomic description and an updated key to the Sindora of Singapore and Peninsular Malaysia are also provided.
... Furthermore, there is a lack of studies on the minimum viable size of tree populations in the conserved areas. Molecular marker studies of genetic diversity, intrapopulation spatial genetic structure (SGS), mating system and pollen/seed dispersal of remaining adult trees of species from before fragmentation and juveniles established after fragmentation help us to determine the evolutionary viability of populations over generations, as well as to estimate the minimum viable area ( MVA ) for in situ conservation (Saccheri and Hanski 2006;Noreen and Webb 2013). ...
Key message
Although all populations show extensive pollen immigration, the occurrence of spatial genetic structure and biparental inbreeding decreased genetic diversity and effective population size.
Abstract
The Brazilian savanna is the second largest Neotropical biome, and a globally important biodiversity hotspot. Basic knowledge of the ecology and genetics of its species can help conserve this important biome. We investigated genetic diversity, spatial genetic structure (SGS), pollen dispersal, and mating system in three Hymenaea stigonocarpa populations (AS, PE, IT) in the Brazilian savanna, using microsatellite loci and samples of adult trees from all populations and seeds from the IT population. As a result of the long geographic distance between populations, the genetic differentiation among them was high (0.397). Individuals of the IT population presented a grouped distribution due to root propagation, resulting in low genotypic richness (GR\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{GR}$$\end{document} = 0.194) and allelic richness (R\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R$$\end{document} = 4), and high SGS (Sp\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{Sp}$$\end{document} = 0.064) compared to AS and PE (GR\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{GR}$$\end{document} > 0.98, R > 5, Sp\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{Sp}$$\end{document} < 0.026) populations. The IT population showed high pollen immigration (46.4%), pollen dispersal distance (up to 3.57 km), and outcrossing rate (0.934–1.0), but matings were correlated (0.01–1), and some occurred among relatives (up to 0.098), resulting in some inbred seeds (0.140), a lower variance effective population size (Ne\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${N}_{e}$$\end{document} = 3.02) than expected with random mating, and an estimate of 50 seed-trees required to retain an Ne\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${N}_{e}$$\end{document} of 150 in samples of maternal progeny. The estimated minimum viable area to retain a reference (Ner\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${N}_{\mathrm{er}}$$\end{document}) of 1000 indicates that the current AS and IT population areas are insufficient to maintain viable populations in the long-term, demonstrating the importance of maintaining surrounding areas for conservation of these H. stigonocarpa populations.
... However, these directional trends of influence on tropical trees' genetic variation are not entirely always constant (Minn et al., 2014;Akinnagbe et al., 2019). For instance, Noreen & Webb (2013) found high genetic diversity in Koompassia malaccensis populations across Singapore despite the high habitat loss. Silvestrini et al. (2015) reported similar levels of genetic diversity of Croton floribundus within some Brazilian primary and successional forests. ...
The global efforts to restore tropical forests and their productive and ecological functions through plantation forestry largely depend on the available genetic variation in the tree species used to establish the plantations. However, there is limited information on the levels and trends of the genetic variation and variability of different plantation tree species in the tropics. Therefore, this study reviews several marker-based studies that have investigated genetic variation. Most of the top economic species like Eucalyptus tereticornis and Mansonia altissima are attributed to low levels of genetic diversity, while others like Pinus caribaea and Swietenia macrophylla still exhibit high expected heterozygosity across different populations. However, the levels of genetic diversity assessed may depend on the markers used. Microsatellites, i.e., simple sequence repeats (SSRs), mostly give higher estimates when compared to other polymerase chain reaction-based markers. Other factors that typically contribute to the directional pattern of genetic variation in tropical tree species and populations include their distribution, density, seed dispersal, succession, and reproduction. Also, anthropogenic impacts like logging and fragmentation have contributed to the vast genetic base reduction of many tropical species and populations. Having adequate genetic variation within the plantation populations is significant in improving their fitness, resilience, fecundity, productivity, and other ecological functions. It also provides a basis for tree improvement and
breeding in plantation forests. Although clonal forestry is becoming widespread and considered highly productive, it is attributed to specific economic, technical, and ecological risks, such as the increased spread of pests and diseases. Therefore, further discussions and recommendations to maximise genetic diversity in tropical (clonal) plantations are provided.
... There have been few genetic studies on the impacts of isolation on plants and animals at Bukit Timah, and possible genetic erosion of their populations. In one of the few population genetic studies of a tree species found at Bukit Timah, Noreen and Webb (2013) looked at genetic diversity and structure in the forest giant Koompassia malaccensis. They found no evidence of a genetic bottleneck nor a reduction in effective population size. ...
Forest fragments are increasingly common in the tropics as pristine forests are cleared. Fragments stranded within cities are especially stress-prone due to their greatly altered environment and high human impacts. We review biodiversity and ecological studies from Bukit Timah, a tropical forest fragment in Singapore, to ask how this forest has contributed to our understanding of tropical ecology and fragmentation effects, and list the conservation values of this forest. Evidence from Amazonian fragments predicts that losses in diversity and forest function follow fragmentation, and although Bukit Timah has adhered to some of those predictions, other aspects of the forest appeared remarkably resilient. As might be expected, declines in plant, invertebrate, bird, and mammal diversity occurred not only historically but also across two surveys made about 20 years apart. In other ways Bukit Timah proved surprising. Aboveground biomass fluctuated but did not plummet drastically, and was comparable to levels found in primary forests in the region. The extirpation of large fauna did not appear to reduce the dispersal of large seeded plant species, likely due to continued dispersal by small-mammals and birds. Exotic tree species are confined to recovering secondary forest fringes and do not threaten the primary forest, except for perhaps shade tolerant Pará rubber and a handful of cultivated fruit trees. Studies of birds and plants found that life history differences could account for differences in genetic connectivity or isolation for different species, with population genetic implications for other taxa. Despite being a small fragment, new species of plants and animals continue to be discovered or rediscovered. Clearly, there are reasons to celebrate Bukit Timah as a forest fragment that withstood two centuries of human impacts. Nonetheless, many measures can be implemented to better secure its future.
... ⎯ D.J. Mabberley (1992: 265) Another area which we know very little about is the population genetics of rainforest plants. Some investigations are throwing light on genetic processes, including how pollen flow (Ng et al., 2006) and seed dispersal (Foster & Sork, 1997) affect genetic differentiation, and the possible influences of life history and ecological traits on levels of genetic diversity (Hamrick et al., 1992;Noreen & Webb, 2013). Whereas selective logging may not significantly reduce the genetic diversity of some canopy tree species (Ang et al., 2017), it appears to do so in a number of cases, where inbreeding with a reduced number of individuals may be a potential outcome (Shorea megistophylla P.S. Ashton: Murawski et al., 1994; Dryobalanops aromatica C.F.Gaertn.: Lee, 2000). ...
Contemporary studies into the spectrum of plant life assembled on the island of Borneo continue to demonstrate an astonishing richness for some groups. Not all lineages are equivalent in their richness, and both biogeographic and ecological factors are the principal correlates of species richness and lineage diversification. The ways in which population genetic factors may influence the generation and persistence of variation, and their interaction with environmental change, could have fundamental importance in how diversity is maintained. Central Sarawak in the northwest Borneo hotspot is a premier ecological theatre where the interplay of such factors operates: its plant species richness is astounding, floristic documentation continues perhaps too slowly, and research and conservation priorities continue to loom large. Unfortunately, this resource has been severely modified in the several decades spanning the turn of the 21st century. The importance of increasing public perception, especially with well-illustrated accounts of this biological richness and its significance through a natural history perspective, will be as critical as the slowly advancing frontiers of the scientific platform on which our understanding depends.
... Other studies in endangered tree species have found high levels of genetic diversity in small fragmented populations, such as for the critically endangered Rhododendron protistum var. giganteum (Forrest ex Tagg) D.F. Chamb. in China (Wu et al. 2015), the locally endangered Koompassia malaccensis Maing. in Singapore (Noreen and Webb 2013), or the locally rare Guaiacum sanctum L. in Costa Rica (Fuchs and Hamrick 2010), which were explained by high levels of historical gene flow. In contrast, low genetic diversity has been found in other endangered species, which was revealed when seedlings and adults were contrasted, such as for the tropical tree Prunus africana (Hook. ...
Magnolias are characteristic tree species of the Tropical Montane Cloud Forest (TMCF) in Mexico, an ecosystem that is highly threatened by habitat fragmentation and climate change. In this study, based on DNA sequences from five regions (chloroplast: trnT-trnL, trnK5-matK, trnS-trnG, rpl32-trnL, nuclear: ITS) and seven nuclear microsatellite markers, we aimed to delineate species boundaries between two-endemic species of the TMCF, Magnolia pedrazae and Magnolia schiedeana, and to estimate levels of genetic structure and diversity among populations. Phylogenetic and haplotype network analyses for the chloroplast and ITS regions did not support genetic differentiation as two distinctive species. Results from Bayesian and multivariate cluster analyses based on microsatellite loci showed high genetic differentiation across most populations, which was consistent with a strong and significant pattern of isolation by geographical distance. We found moderate to high levels of population genetic diversity, but it was lower in small populations relative to large populations. Our results suggest a contemporary decrease of genetic connectivity among populations, likely as a consequence of the current decline of suit-able TMCF habitat. Managing landscape connectivity among remnant Magnolia populations within protected natural parks and surroundings, and with emphasis of small populations, would be key for the species conservation.
Link to read the pdf: https://rdcu.be/bh0qY
... Our analyses revealed high levels of genetic diversity within populations of Senegalia senegal. Mean expected heterozygosity (H E = 0.56; Table 2) found in this study is comparable to values found in other microsatellite studies of Senegalia species (S. senegal: H E = 0.67, [7]; H E = 0.53, [72]; S. dudgeoni, H E = 0.6 [72]; S. tortilis, H E = 0.7, [73]) and other tropical tree species, including Milicia excelsa (H E = 0.81, [74]), Koompas- sia malaccensis (H E = 0.85, [75]), and Vitellaria paradoxa (H E = 0.73, [76]). Conversely, low levels of genetic diversity can lead to inbreeding depression in the short-term, and to reduced evolutionary potential in the longer term [70,77]. ...
Climate change is predicted to impact species’ genetic diversity and distribution. We used Senegalia senegal (L.) Britton, an economically important species distributed in the Sudano-Sahelian savannah belt of West Africa, to investigate the impact of climate change on intraspecific genetic diversity and distribution. We used ten nuclear and two plastid microsatellite markers to assess genetic variation, population structure and differentiation across thirteen sites in West Africa. We projected suitable range, and potential impact of climate change on genetic diversity using a maximum entropy approach, under four different climate change scenarios. We found higher genetic and haplotype diversity at both nuclear and plastid markers than previously reported. Genetic differentiation was strong for chloroplast and moderate for the nuclear genome. Both genomes indicated three spatially structured genetic groups. The distribution of Senegalia senegal is strongly correlated with extractable nitrogen, coarse fragments, soil organic carbon stock, precipitation of warmest and coldest quarter and mean temperature of driest quarter. We predicted 40.96 to 6.34 per cent of the current distribution to favourably support the species’ ecological requirements under future climate scenarios. Our results suggest that climate change is going to affect the population genetic structure of Senegalia senegal, and that patterns of genetic diversity are going to influence the species’ adaptive response to climate change. Our study contributes to the growing evidence predicting the loss of economically relevant plants in West Africa in the next decades due to climate change.
... Previous studies of polymorphism at putatively neutral loci have shown that tropical trees tend to have reduced genetic variation in small local populations (Hamrick & Murawski 1991;Finkeldey & Hattemer 2007;Noreen & Webb 2013) and strongly increased seedling-to-adult survival for outcrossed progeny (Hufford & Hamrick 2003). Over a scale of a few tens of metres, tropical trees tend to be related, corresponding with seed dispersal kernels, with most pollen originating within~100 to 1000 m of the mother tree, and detectable levels of long-distance pollen flow (Loveless 1992;Boshier et al. 1995;Hardy et al. 2006;Noreen & Webb 2013). ...
... Previous studies of polymorphism at putatively neutral loci have shown that tropical trees tend to have reduced genetic variation in small local populations (Hamrick & Murawski 1991;Finkeldey & Hattemer 2007;Noreen & Webb 2013) and strongly increased seedling-to-adult survival for outcrossed progeny (Hufford & Hamrick 2003). Over a scale of a few tens of metres, tropical trees tend to be related, corresponding with seed dispersal kernels, with most pollen originating within~100 to 1000 m of the mother tree, and detectable levels of long-distance pollen flow (Loveless 1992;Boshier et al. 1995;Hardy et al. 2006;Noreen & Webb 2013). ...
In tropical forests, rarer species show increased sensitivity to species-specific soil pathogens and more negative effects of conspecific density on seedling survival (NDD). These patterns suggest a connection between ecology and immunity, perhaps because small population size disproportionately reduces genetic diversity of hyperdiverse loci such as immunity genes. In an experiment examining seedling roots from six species in one tropical tree community, we found that smaller populations have reduced amino acid diversity in pathogen resistance (R) genes but not the transcriptome in general. Normalized R gene amino acid diversity varied with local abundance and prior measures of differences in sensitivity to conspecific soil and NDD. After exposure to live soil, species with lower R gene diversity had reduced defense gene induction, more co-susceptibility within maternal cohorts to colonization by potentially pathogenic fungi, reduced root growth arrest (an R gene mediated response), and their root-associated fungi showed lower induction of self-defense (anti-oxidants). Local abundance was not related to the ability to induce immune responses when pathogen recognition was bypassed by application of salicylic acid, a phytohormone that activates defense responses downstream of R gene signaling. These initial results support the hypothesis that smaller local tree populations have reduced R gene diversity and recognition-dependent immune responses, along with greater co-susceptibility to species-specific pathogens that may facilitate disease transmission and NDD. Locally rare species may be less able to increase their equilibrium abundance without genetic boosts to defense via immigration of novel R gene alleles from a larger and more diverse regional population. This article is protected by copyright. All rights reserved.
