Intraspecific trait variation influences physiological performance and fitness in the South Africa shrub genus Protea (Proteaceae).

BACKGROUND AND AIMS: Global plant trait datasets commonly identify trait relationships that are interpreted to reflect fundamental trade-offs associated with plant strategies, but often these trait relationships are not identified when evaluating them at smaller taxonomic and spatial scales. In this study we evaluate trait relationships measured on individual plants for five widespread Protea species in South Africa to determine whether broad-scale patterns of structural trait (e.g. leaf area) and physiological trait (e.g. photosynthetic rates) relationships can be detected within natural populations, and if these traits are themselves related to plant fitness. METHODS: We evaluated the variance structure (i.e. the proportional intraspecific trait variation relative to among-species variation) for nine structural traits and six physiological traits measured in wild populations. We used a multivariate path model to evaluate the relationships between structural traits and physiological traits, and the relationship between these traits and plant size and reproductive effort. KEY RESULTS: While intraspecific trait variation is relatively low for structural traits, it accounts for between 50 and 100 % of the variation in physiological traits. Furthermore, we identified few trait associations between any one structural trait and physiological trait, but multivariate regressions revealed clear associations between combinations of structural traits and physiological performance (R2 = 0.37-0.64), and almost all traits had detectable associations with plant fitness. CONCLUSIONS: Intraspecific variation in structural traits leads to predictable differences in individual-level physiological performance in a multivariate framework, even though the relationship of any particular structural trait to physiological performance may be weak or undetectable. Furthermore, intraspecific variation in both structural and physiological traits leads to differences in plant size and fitness. These results demonstrate the importance of considering measurements of multivariate phenotypes on individual plants when evaluating trait relationships and how trait variation influences predictions of ecological and evolutionary outcomes.

Genetic structure in patchy populations of a candidate foundation plant: a case study of Leymus chinensis using genetic and clonal diversity.

PREMISE: The distribution of genetic diversity on the landscape has critical ecological and evolutionary implications. This may be especially the case on a local scale for foundation plant species because they create and define ecological communities, contributing disproportionately to ecosystem function. METHODS: We examined the distribution of genetic diversity and clones, which we defined first as unique multilocus genotypes (MLG), and then by grouping similar MLGs into multilocus lineages. We used 186 markers from inter-simple sequence repeats (ISSR) across 358 ramets from 13 patches of the foundation grass Leymus chinensis. We examined the relationship between genetic and clonal diversities, their variation with patch size, and the effect of the number of markers used to evaluate genetic diversity and structure in this species. RESULTS: Every ramet had a unique MLG. Almost all patches consisted of individuals belonging to a single multilocus lineages. We confirmed this with a clustering algorithm to group related genotypes. The predominance of a single lineage within each patch could be the result of the accumulation of somatic mutations, limited dispersal, some sexual reproduction with partners mainly restricted to the same patch, or a combination of all three. CONCLUSIONS: We found strong genetic structure among patches of L. chinensis. Consistent with previous work on the species, the clustering of similar genotypes within patches suggests that clonal reproduction combined with somatic mutation, limited dispersal, and some degree of sexual reproduction among neighbors causes individuals within a patch to be more closely related than among patches.

Microscale trait-environment associations in two closely-related South African shrubs.

PREMISE OF THE STUDY: Plant traits are often associated with the environments in which they occur, but these associations often differ across spatial and phylogenetic scales. Here we study the relationship between microenvironment, microgeographical location, and traits within populations using co-occurring populations of two closely related evergreen shrubs in the genus Protea. METHODS: We measured a suite of functional traits on 147 plants along a single steep mountainside where both species occur, and we used data-loggers and soil analyses to characterize the environment at 10 microsites spanning the elevational gradient. We used Bayesian path analyses to detect trait-environment relationships in the field for each species. We used complementary data from greenhouse grown seedlings derived from wild collected seed to determine whether associations detected in the field are the result of genetic differentiation. KEY RESULTS: Microenvironmental variables differed substantially across our study site. We found strong evidence for six trait-environment associations, although these differed between species. We were unable to detect similar associations in greenhouse-grown seedlings. CONCLUSIONS: Several leaf traits were associated with temperature and soil variation in the field, but the inability to detect these in the greenhouse suggests that differences in the field are not the result of genetic differentiation.

Anchored phylogenomics improves the resolution of evolutionary relationships in the rapid radiation of Protea L.

PREMISE OF THE STUDY: Estimating phylogenetic relationships in relatively recent evolutionary radiations is challenging, especially if short branches associated with recent divergence result in multiple gene tree histories. We combine anchored enrichment next-generation sequencing with species tree analyses to produce a robust estimate of phylogenetic relationships in the genus Protea (Proteaceae), an iconic radiation in South Africa. METHODS: We sampled multiple individuals within 59 out of 112 species of Protea and 6 outgroup species for a total of 163 individuals, and obtained sequences for 498 low-copy, orthologous nuclear loci using anchored phylogenomics. We compare several approaches for building species trees, and explore gene tree-species tree discrepancies to determine whether poor phylogenetic resolution reflects a lack of informative sites, incomplete lineage sorting, or hybridization. KEY RESULTS: Phylogenetic estimates from species tree approaches are similar to one another and recover previously well-supported clades within Protea, in addition to providing well-supported phylogenetic hypotheses for previously poorly resolved intrageneric relationships. Individual gene trees are markedly different from one another and from species trees. Nonetheless, analyses indicate that differences among gene trees occur primarily concerning clades supported by short branches. CONCLUSIONS: Species tree methods using hundreds of nuclear loci provided strong support for many previously unresolved relationships in the radiation of the genus Protea. In cases where support for particular relationships remains low, these appear to arise from few informative sites and lack of information rather than strongly supported disagreement among gene trees.

Isolation by distance and isolation by environment contribute to population differentiation in Protea repens (Proteaceae L.), a widespread South African species.

PREMISE OF THE STUDY: The Cape Floristic Region (CFR) of South Africa is renowned for its botanical diversity, but the evolutionary origins of this diversity remain controversial. Both neutral and adaptive processes have been implicated in driving diversification, but population-level studies of plants in the CFR are rare. Here, we investigate the limits to gene flow and potential environmental drivers of selection in Protea repens L. (Proteaceae L.), a widespread CFR species. METHODS: We sampled 19 populations across the range of P. repens and used genotyping by sequencing to identify 2066 polymorphic loci in 663 individuals. We used a Bayesian FST outlier analysis to identify single-nucleotide polymorphisms (SNPs) marking genomic regions that may be under selection; we used those SNPs to identify potential drivers of selection and excluded them from analyses of gene flow and genetic structure. RESULTS: A pattern of isolation by distance suggested limited gene flow between nearby populations. The populations of P. repens fell naturally into two or three groupings, which corresponded to an east-west split. Differences in rainfall seasonality contributed to diversification in highly divergent loci, as do barriers to gene flow that have been identified in other species. CONCLUSIONS: The strong pattern of isolation by distance is in contrast to the findings in the only other widespread species in the CFR that has been similarly studied, while the effects of rainfall seasonality are consistent with well-known patterns. Assessing the generality of these results will require investigations of other CFR species.

Transcriptome sequencing reveals population differentiation in gene expression linked to functional traits and environmental gradients in the South African shrub Protea repens.

Understanding the environmental and genetic mechanisms underlying locally adaptive trait variation across the ranges of species is a major focus of evolutionary biology. Combining transcriptome sequencing with common garden experiments on populations spanning geographical and environmental gradients holds promise for identifying such mechanisms. The South African shrub Protea repens displays diverse phenotypes in the wild along drought and temperature gradients. We grew plants from seeds collected at 19 populations spanning this species' range, and sequenced the transcriptomes of these plants to reveal gene pathways associated with adaptive trait variation. We related expression in co-expressed gene networks to trait phenotypes measured in the common garden and to source population climate. We found that expression in gene networks correlated with source-population environment and with plant traits. In particular, the activity of gene networks enriched for growth related pathways correlated strongly with source site minimum winter temperature and with leaf size, stem diameter and height in the garden. Other gene networks with enrichments for photosynthesis related genes showed associations with precipitation. Our results strongly suggest that this species displays population-level differences in gene expression that have been shaped by source population site climate, and that are reflected in trait variation along environmental gradients.

Intraspecific variation in stomatal traits, leaf traits and physiology reflects adaptation along aridity gradients in a South African shrub.

BACKGROUND AND AIMS: Trait-environment relationships are commonly interpreted as evidence for local adaptation in plants. However, even when selection analyses support this interpretation, the mechanisms underlying differential benefits are often unknown. This study addresses this gap in knowledge using the broadly distributed South African shrub Protea repens. Specifically, the study examines whether broad-scale patterns of trait variation are consistent with spatial differences in selection and ecophysiology in the wild. METHODS: In a common garden study of plants sourced from 19 populations, associations were measured between five morphological traits and three axes describing source climates. Trait-trait and trait-environment associations were analysed in a multi-response model. Within two focal populations in the wild, selection and path analyses were used to test associations between traits, fecundity and physiological performance. KEY RESULTS: Across 19 populations in a common garden, stomatal density increased with the source population's mean annual temperature and decreased with its average amount of rainfall in midsummer. Concordantly, selection analysis in two natural populations revealed positive selection on stomatal density at the hotter, drier site, while failing to detect selection at the cooler, moister site. Dry-site plants with high stomatal density also had higher stomatal conductances, cooler leaf temperatures and higher light-saturated photosynthetic rates than those with low stomatal density, but no such relationships were present among wet-site plants. Leaf area, stomatal pore index and specific leaf area in the garden also co-varied with climate, but within-population differences were not associated with fitness in either wild population. CONCLUSIONS: The parallel patterns of broad-scale variation, differences in selection and differences in trait-ecophysiology relationships suggest a mechanism for adaptive differentiation in stomatal density. Densely packed stomata may improve performance by increasing transpiration and cooling, but predominately in drier, hotter climates. This study uniquely shows context-dependent benefits of stomatal density--a trait rarely linked to local adaptation in plants.

Functional Traits in Parallel Evolutionary Radiations and Trait-Environment Associations in the Cape Floristic Region of South Africa.

Evolutionary radiations with extreme levels of diversity present a unique opportunity to study the role of the environment in plant evolution. If environmental adaptation played an important role in such radiations, we expect to find associations between functional traits and key climatic variables. Similar trait-environment associations across clades may reflect common responses, while contradictory associations may suggest lineage-specific adaptations. Here, we explore trait-environment relationships in two evolutionary radiations in the fynbos biome of the highly biodiverse Cape Floristic Region (CFR) of South Africa. Protea and Pelargonium are morphologically and evolutionarily diverse genera that typify the CFR yet are substantially different in growth form and morphology. Our analytical approach employs a Bayesian multiple-response generalized linear mixed-effects model, taking into account covariation among traits and controlling for phylogenetic relationships. Of the pairwise trait-environment associations tested, 6 out of 24 were in the same direction and 2 out of 24 were in opposite directions, with the latter apparently reflecting alternative life-history strategies. These findings demonstrate that trait diversity within two plant lineages may reflect both parallel and idiosyncratic responses to the environment, rather than all taxa conforming to a global-scale pattern. Such insights are essential for understanding how trait-environment associations arise and how they influence species diversification.

Extrapolating from local ecological processes to genus-wide patterns in colour polymorphism in South African Protea.

Polymorphic traits are central to many fundamental discoveries in evolution, yet why they are found in some species and not others remains poorly understood. We use the African genus Protea-within which more than 40% of species have co-occurring pink and white floral colour morphs-to ask whether convergent evolution and ecological similarity could explain the genus-wide pattern of polymorphism. First, we identified environmental correlates of pink morph frequency across 28 populations of four species. Second, we determined whether the same correlates could predict species-level polymorphism and monomorphism across 31 species. We found that pink morph frequency increased with elevation in Protea repens and three section Exsertae species, increased eastward in P. repens, and increased with seed predation intensity in section Exsertae. For cross-species comparisons, populations of monomorphic pink species occurred at higher elevations than populations of monomorphic white species, and 18 polymorphic species spanned broader elevational gradients than 13 monomorphic species. These results suggest that divergent selection along elevational clines has repeatedly favoured polymorphism, and that more uniform selection in altitudinally restricted species may promote colour monomorphism. Our findings are, to our knowledge, the first to link selection acting within species to the presence and absence of colour polymorphism at broader phylogenetic scales.

Genetics in geographically structured populations: defining, estimating and interpreting F(ST).

Wright's F-statistics, and especially F(ST), provide important insights into the evolutionary processes that influence the structure of genetic variation within and among populations, and they are among the most widely used descriptive statistics in population and evolutionary genetics. Estimates of F(ST) can identify regions of the genome that have been the target of selection, and comparisons of F(ST) from different parts of the genome can provide insights into the demographic history of populations. For these reasons and others, F(ST) has a central role in population and evolutionary genetics and has wide applications in fields that range from disease association mapping to forensic science. This Review clarifies how F(ST) is defined, how it should be estimated, how it is related to similar statistics and how estimates of F(ST) should be interpreted.

Direct and indirect selection on floral pigmentation by pollinators and seed predators in a color polymorphic South African shrub.

The coexistence of different color morphs is often attributed to variable selection pressures across space, time, morph frequencies, or selection agents, but the routes by which each morph is favored are rarely identified. In this study we investigated factors that influence floral color polymorphisms on a local scale in Protea, within which approximately 40% of species are polymorphic. Previous work shows that seed predators and reproductive differences likely contribute to maintaining polymorphism in four Protea species. We explored whether selection acts directly or indirectly on floral color in two populations of Protea aurea, using path analysis of pollinator behavior, nectar production, seed predation, color, morphology, and maternal fecundity fitness components. We found that avian pollinators spent more time on white morphs, likely due to nectar differences, but that this had no apparent consequences for fecundity. Instead, the number of flowers per inflorescence underpinned many of the reproductively important differences between color morphs. White morphs had more flowers per inflorescence, which itself was positively correlated with nectar production, seed predator occurrence, and total long-term seed production. The number of seeds per plant to survive predation, in contrast, was not directly associated with color or any other floral trait. Thus, although color differences may be associated with conflicting selection pressures, the selection appears to be associated with the number of flowers per inflorescence and its unmeasured correlates, rather than with inflorescence color itself.

Estimating covariation between vital rates: a simulation study of connected vs. separate generalized linear mixed models (GLMMs).

Covariation between vital rates is recognized as an important pattern to be accounted for in demographic modeling. We recently introduced a model for estimating vital rates and their covariation as a function of known and unknown effects, using generalized linear mixed models (GLMM's) implemented in a hierarchical Bayesian framework (Evans et al., 2010) In particular, this model included a model-wide year effect (YEAR) influencing all vital rates, which we used to estimate covariation between vital rates due to exogenous factors not directly included in the model. This YEAR effect connected the GLMMs of vital rates into one large model; we refer to this as the "connected GLMMs" approach. Here we used a simulation study to evaluate the performance of a simplified version of this model, compared to separate GLMMs of vital rates, in terms of their ability to estimate correlations between vital rates. We simulated data from known relationships between vital rates and a covariate, inducing correlations among the vital rates. We then estimated those correlations from the simulated data using connected vs. separate GLMMs with year random effects. We compared precision and accuracy of estimated vital rates and their correlations under three scenarios of the pervasiveness of the exogenous effect (and thus true correlations). The two approaches provide equally good point estimates of vital rate parameters, but connected GLMMs provide better estimates of covariation between vital rates than separate GLMMs, both in terms of accuracy and precision, when the common influence on vital rates is pervasive. We discuss the situations where connected GLMMs might be best used, as well as further areas of investigation for this approach.

Extreme environments select for reproductive assurance: evidence from evening primroses (Oenothera).

Competing evolutionary forces shape plant breeding systems (e.g. inbreeding depression, reproductive assurance). Which of these forces prevails in a given population or species is predicted to depend upon such factors as life history, ecological conditions, and geographical context. Here, we examined two such predictions: that self-compatibility should be associated with the annual life history or extreme climatic conditions. We analyzed data from a clade of plants remarkable for variation in breeding system, life history and climatic conditions (Oenothera, sections Anogra and Kleinia, Onagraceae). We used a phylogenetic comparative approach and Bayesian or hybrid Bayesian tests to account for phylogenetic uncertainty. Geographic information system (GIS)-based climate data and ecological niche modeling allowed us to quantify climatic conditions. Breeding system and reproductive life span are not correlated in Anogra and Kleinia. Instead, self-compatibility is associated with the extremes of temperature in the coldest part of the year and precipitation in the driest part of the year. In the 60 yr since this pattern was anticipated, this is the first demonstration of a relationship between the evolution of self-compatibility and climatic extremes. We discuss possible explanations for this pattern and possible implications with respect to anthropogenic climate change.

Genetic diversity of microsatellite loci in hierarchically structured populations.

Microsatellite loci are widely used for investigating patterns of genetic variation within and among populations. Those patterns are in turn determined by population sizes, migration rates, and mutation rates. We provide exact expressions for the first two moments of the allele frequency distribution in a stochastic model appropriate for studying microsatellite evolution with migration, mutation, and drift under the assumption that the range of allele sizes is bounded. Using these results, we study the behavior of several measures related to Wright's F(ST), including Slatkin's R(ST). Our analytical approximations for F(ST) and R(ST) show that familiar relationships between N(e)m and F(ST) or R(ST) hold when the migration and mutation rates are small. Using the exact expressions for F(ST) and R(ST), our numerical results show that, when the migration and mutation rates are large, these relationships no longer hold. Our numerical results also show that the diversity measures most closely related to F(ST) depend on mutation rates, mutational models (stepwise versus two-phase), migration rates, and population sizes. Surprisingly, R(ST) is relatively insensitive to the mutation rates and mutational models. The differing behaviors of R(ST) and F(ST) suggest that properties of the among-population distribution of allele frequencies may allow the roles of mutation and migration in producing patterns of diversity to be distinguished, a topic of continuing investigation.

A Beta-mixture model for assessing genetic population structure.

An important fraction of recently generated molecular data is dominant markers. They contain substantial information about genetic variation but dominance makes it impossible to apply standard techniques to calculate measures of genetic differentiation, such as F-statistics. In this article, we propose a new Bayesian beta-mixture model that more accurately describes the genetic structure from dominant markers and estimates multiple F(ST) s from the sample. The model also has important application for codominant markers and single-nucleotide polymorphism (SNP) data. The number of F(ST) is assumed unknown beforehand and follows a random distribution. The reversible jump algorithm is used to estimate the unknown number of multiple F(ST) s. We evaluate the performance of three split proposals and the overall performance of the proposed model based on simulated dominant marker data. The model could reliably identify and estimate a spectrum of degrees of genetic differentiation present in multiple loci. The estimates of F(ST) s also incorporate uncertainty about the magnitude of within-population inbreeding coefficient. We illustrate the method with two examples, one using dominant marker data from a rare orchid and the other using codominant marker data from human populations.

Nitrogen deposition, competition and the decline of a regionally threatened legume, Desmodium cuspidatum.

Increased nitrogen (N) deposition, resulting from the combustion of fossil fuels, production of synthetic fertilizers, growth of N(2)-fixing crops and high-intensity agriculture, is one of the anthropogenic factors most likely to cause global biodiversity changes over the next century. This influence may be especially large in temperate zone forests, which are highly N limited and occur in regions with the highest levels of N deposition. Within these ecosystems, N(2)-fixing plants, including legumes, may be more sensitive to N deposition than other plant species. Though it has long been recognized that the competitive edge conferred by N(2)-fixation diminishes with increasing soil N availability, the conservation implications of increased N deposition on native N(2)-fixers have received less attention. We focus on Desmodium cuspidatum, which has experienced dramatic population losses in the last 30-40 years in the northeastern United States. We explore competition between this regionally threatened legume and a common non-N(2)-fixing neighbor, Solidago canadensis, across a gradient of N deposition. Our data show that increased N deposition may be detrimental to N(2)-fixers such as D. cuspidatum in two ways: (1) biomass accumulation in the non-N(2)-fixer, S. canadensis, responds more strongly to increasing N deposition, and (2) S. canadensis competes strongly for available mineral nitrogen and can assimilate N previously fixed by D. cuspidatum, resulting in D. cuspidatum relying more heavily on energetically expensive N(2)-fixation when grown with S. canadensis. N deposition may thus reduce or eliminate the competitive advantage of N(2)-fixing species growing in N-limited ecosystems.

Was it an explosion? Using population genetics to explore the dynamics of a recent radiation within Protea (Proteaceae L.).

Adaptive radiations likely underlie much of the world's diversity, especially that of hyper-diverse regions. They are usually characterized by a burst of speciation early in their evolutionary history, a pattern which can be detected using population genetic tools. The Cape Floristic Region (CFR) of southwestern South Africa is home to many spectacular plant radiations. Here, we investigate the white proteas (Protea section Exsertae), a typical CFR radiation, to determine if it demonstrates the burst of speciation associated with adaptive radiations in recent models. Inferences from individual assignment, tree-based population relationships, and pairwise F-statistics based on 10 microsatellite loci reveal that while the white proteas radiated recently they did not radiate explosively. In addition, we found evidence that there is little gene flow between sampled populations of most species. Taken together, these results demonstrate that within a small clade, the processes underlying the radiation are different from those envisioned by current models of adaptive radiation and suggest that geographical isolation could have played a role in the diversification of the group. Our study implicates both adaptive and non-adaptive processes in the evolution of botanical diversity of the CFR.

Proximity is not a proxy for parentage in an animal-dispersed Neotropical canopy palm.

We used parentage analysis to estimate seedling recruitment distances and genetic composition of seedling patches centred around reproductive trees of the animal-dispersed Neotropical canopy palm Iriartea deltoidea in two 0.5 ha plots within second-growth forest and one 0.5 ha plot in adjacent old-growth forest at La Selva Biological Field Station in north-eastern Costa Rica. Seedlings were significantly spatially aggregated in all plots, but this pattern was not due to dispersal limitation. More than 70 per cent of seedlings were dispersed at least 50 m from parent trees. Few seedlings were offspring of the closest reproductive trees. Seedling patches observed beneath reproductive trees originate from dozens of parental trees. Observed patterns of seedling distribution and spatial genetic structure are largely determined by the behaviour of vertebrate seed dispersers rather than by spatial proximity to parental trees.

Correlated evolution between climate and suites of traits along a fast-slow continuum in the radiation of Protea.

Evolutionary radiations are responsible for much of Earth's diversity, yet the causes of these radiations are often elusive. Determining the relative roles of adaptation and geographic isolation in diversification is vital to understanding the causes of any radiation, and whether a radiation may be labeled as "adaptive" or not. Across many groups of plants, trait-climate relationships suggest that traits are an important indicator of how plants adapt to different climates. In particular, analyses of plant functional traits in global databases suggest that there is an "economics spectrum" along which combinations of functional traits covary along a fast-slow continuum. We examine evolutionary associations among traits and between trait and climate variables on a strongly supported phylogeny in the iconic plant genus Protea to identify correlated evolution of functional traits and the climatic-niches that species occupy. Results indicate that trait diversification in Protea has climate associations along two axes of variation: correlated evolution of plant size with temperature and leaf investment with rainfall. Evidence suggests that traits and climatic-niches evolve in similar ways, although some of these associations are inconsistent with global patterns on a broader phylogenetic scale. When combined with previous experimental work suggesting that trait-climate associations are adaptive in Protea, the results presented here suggest that trait diversification in this radiation is adaptive.

Multigenerational genetic analysis of tropical secondary regeneration in a canopy palm.

Iriartea deltoidea (Arecaceae) is an abundant canopy palm with a wide geographic distribution in Neotropical wet forests. We analyzed the genetic profile across three generations of Iriartea within a 43-ha area encompassing two areas of second-growth and adjoining old-growth forest at La Selva Biological Field Station in northeastern Costa Rica. A total of 311 reproductively mature trees, 99 large saplings, 207 small saplings, and 601 seedlings were genotyped using 141 AFLP loci. Parentage analysis revealed high dispersal distances, both for seed (over 2.3 km) and pollen (over 3.8 km), indicating a large genetic neighborhood within La Selva Biological Station. In a 20-ha area of second growth, the founding palm population was dominated by a small number of parental trees located in the adjacent old-growth forest; two old-growth trees contributed 48% of the second-growth genes. The genetic diversity of reproductively mature trees in this second-growth forest was significantly reduced compared to adjacent old-growth forest. Within 400 m of the border with old-growth forest, we observed a similar reduction of genetic diversity in saplings, and an even greater loss of genetic diversity in the second generation of seedlings. Nearly half of these seedlings were offspring of local parents. In contrast, in the distant portion of second-growth forest (400-800 m from the old-growth border), parentage analysis showed that 40% of seedlings originated from outside the study area and only 10% were offspring of local parents. These high levels of gene flow maintained genetic diversity in saplings and seedlings similar to levels observed in old-growth forest. Our findings highlight the importance of gene flow from diverse seed and pollen sources for sustaining levels of genetic diversity of tree populations in second-growth forests.