Cumulative nitrogen enrichment alters the drivers of grassland overyielding.

Effects of plant diversity on grassland productivity, or overyielding, are found to be robust to nutrient enrichment. However, the impact of cumulative nitrogen (N) addition (total N added over time) on overyielding and its drivers are underexplored. Synthesizing data from 15 multi-year grassland biodiversity experiments with N addition, we found that N addition decreases complementarity effects and increases selection effects proportionately, resulting in no overall change in overyielding regardless of N addition rate. However, we observed a convex relationship between overyielding and cumulative N addition, driven by a shift from complementarity to selection effects. This shift suggests diminishing positive interactions and an increasing contribution of a few dominant species with increasing N accumulation. Recognizing the importance of cumulative N addition is vital for understanding its impacts on grassland overyielding, contributing essential insights for biodiversity conservation and ecosystem resilience in the face of increasing N deposition.

Effects of plant diversity on productivity strengthen over time due to trait-dependent shifts in species overyielding.

Plant diversity effects on community productivity often increase over time. Whether the strengthening of diversity effects is caused by temporal shifts in species-level overyielding (i.e., higher species-level productivity in diverse communities compared with monocultures) remains unclear. Here, using data from 65 grassland and forest biodiversity experiments, we show that the temporal strength of diversity effects at the community scale is underpinned by temporal changes in the species that yield. These temporal trends of species-level overyielding are shaped by plant ecological strategies, which can be quantitatively delimited by functional traits. In grasslands, the temporal strengthening of biodiversity effects on community productivity was associated with increasing biomass overyielding of resource-conservative species increasing over time, and with overyielding of species characterized by fast resource acquisition either decreasing or increasing. In forests, temporal trends in species overyielding differ when considering above- versus belowground resource acquisition strategies. Overyielding in stem growth decreased for species with high light capture capacity but increased for those with high soil resource acquisition capacity. Our results imply that a diversity of species with different, and potentially complementary, ecological strategies is beneficial for maintaining community productivity over time in both grassland and forest ecosystems.

Evaluation of seed-dispersal services by ants at a temperate pasture: Results of direct observations in an ant suppression experiment.

Ants disperse seeds of many plant species adapted to myrmecochory. While advantages of this ant-plant mutualism for myrmecochorous plants (myrmecochores) have been previously studied in temperate region mostly in forests, our study system was a pasture. Moreover, we used a unique combination of observing the effect of ant-activity suppression on ant dispersal and comparison of the contribution of ant and unassisted dispersal to the distance from mother plant. We established plots without and with ant-activity suppression (enclosures). We offered diaspores of a myrmecochorous (Knautia arvensis), and a non-myrmecochorous (Plantago lanceolata) species in a choice test and followed ants carrying diaspores during days and nights (focus of previous studies was on diurnal dispersal). We measured frequency and distances of ant dispersal and compared them with unassisted dispersal recorded using sticky trap method. The dispersal frequency was lower in enclosures (3.16 times). Ants strongly preferred diaspores of the myrmecochore to non-myrmecochore with 586 and 42 dispersal events, respectively (out of 6400 diaspores of each species offered). Ant dispersal resulted in more even and on average longer distances (maximum almost tenfold longer, 994 cm) in comparison to unassisted dispersal. Ant dispersal altered the distribution of distances of the myrmecochore from roughly symmetric for unassisted dispersal to positively skewed. Ants dispersed heavier diaspores farther. Ants dropped the majority of diaspores during the dispersal (which reduces clustering of seeds), while several (11%) were carried into anthills. Anthills are disturbed microsites presumably favorable for germination in competitive habitats. Ants provided non-negligible dispersal services to myrmecochorous K. arvensis but also, to a lesser extent, of non-myrmecochorous P. lanceolata.

Functional trait trade-offs define plant population stability across different biomes.

Ecological theory posits that temporal stability patterns in plant populations are associated with differences in species' ecological strategies. However, empirical evidence is lacking about which traits, or trade-offs, underlie species stability, especially across different biomes. We compiled a worldwide collection of long-term permanent vegetation records (greater than 7000 plots from 78 datasets) from a large range of habitats which we combined with existing trait databases. We tested whether the observed inter-annual variability in species abundance (coefficient of variation) was related to multiple individual traits. We found that populations with greater leaf dry matter content and seed mass were more stable over time. Despite the variability explained by these traits being low, their effect was consistent across different datasets. Other traits played a significant, albeit weaker, role in species stability, and the inclusion of multi-variate axes or phylogeny did not substantially modify nor improve predictions. These results provide empirical evidence and highlight the relevance of specific ecological trade-offs, i.e. in different resource-use and dispersal strategies, for plant populations stability across multiple biomes. Further research is, however, necessary to integrate and evaluate the role of other specific traits, often not available in databases, and intraspecific trait variability in modulating species stability.

The functional structure of plant communities drives soil functioning via changes in soil abiotic properties.

While biodiversity is expected to enhance multiple ecosystem functions (EFs), the different roles of multiple biodiversity dimensions remain difficult to disentangle without carefully designed experiments. We sowed plant communities with independent levels of functional (FD) and phylogenetic diversities (PD), combined with different levels of fertilization, to investigate their direct and indirect roles on multiple EFs, including plant-related EFs (plant biomass productivity, litter decomposability), soil fertility (organic carbon and nutrient pool variables), soil microbial activity (respiration and nutrient cycling), and an overall multifunctionality. We expected an increase in most EFs in communities with higher values of FD and/or PD via complementarity effects, but also the dominant plant types (using community weighted mean, CWM, independent of FD and PD) via selection effects on several EFs. The results showed strong direct effects of different dimensions of plant functional structure parameters on plant-related EFs, through either CWM or FD, with weak effects of PD. Fertilization had significant effects on one soil microbial activity and indirect effects on the other variables via changes in soil abiotic properties. Dominant plant types and FD showed only indirect effects on soil microbial activity, through litter decomposition and soil abiotic properties, highlighting the importance of cascading effects. This study shows the relevance of complementary dimensions of biodiversity for assessing both direct and cascading effects on multiple EFs.

Common spatial patterns of trees in various tropical forests: Small trees are associated with increased diversity at small spatial scales.

Tropical forests are notable for their high species diversity, even on small spatial scales, and right-skewed species and size abundance distributions. The role of individual species as drivers of the spatial organization of diversity in these forests has been explained by several hypotheses and processes, for example, stochastic dilution, negative density dependence, or gap dynamics. These processes leave a signature in spatial distribution of small trees, particularly in the vicinity of large trees, likely having stronger effects on their neighbors. We are exploring species diversity patterns within the framework of various diversity-generating hypotheses using individual species-area relationships. We used the data from three tropical forest plots (Wanang-Papua New Guinea, Barro Colorado Island-Panama, and Sinharaja-Sri Lanka) and included also the saplings (DBH ≥ 1 cm). Resulting cross-size patterns of species richness and evenness reflect the dynamics of saplings affected by the distribution of large trees. When all individuals with DBH ≥1 cm are included, ~50% of all tree species from the 25- or 50-ha plot can be found within 35 m radius of an individual tree. For all trees, 72%-78% of species were identified as species richness accumulators, having more species present in their surroundings than expected by null models. This pattern was driven by small trees as the analysis of DBH >10 cm trees showed much lower proportion of accumulators, 14%-65% of species identified as richness repellers and had low richness of surrounding small trees. Only 11%-26% of species had lower species evenness than was expected by null models. High proportions of species richness accumulators were probably due to gap dynamics and support Janzen-Connell hypothesis driven by competition or top-down control by pathogens and herbivores. Observed species diversity patterns show the importance of including small tree size classes in analyses of the spatial organization of diversity.

Functional trait effects on ecosystem stability: assembling the jigsaw puzzle.

Under global change, how biological diversity and ecosystem services are maintained in time is a fundamental question. Ecologists have long argued about multiple mechanisms by which local biodiversity might control the temporal stability of ecosystem properties. Accumulating theories and empirical evidence suggest that, together with different population and community parameters, these mechanisms largely operate through differences in functional traits among organisms. We review potential trait-stability mechanisms together with underlying tests and associated metrics. We identify various trait-based components, each accounting for different stability mechanisms, that contribute to buffering, or propagating, the effect of environmental fluctuations on ecosystem functioning. This comprehensive picture, obtained by combining different puzzle pieces of trait-stability effects, will guide future empirical and modeling investigations.

Biotic homogenization destabilizes ecosystem functioning by decreasing spatial asynchrony.

Our planet is facing significant changes of biodiversity across spatial scales. Although the negative effects of local biodiversity (α diversity) loss on ecosystem stability are well documented, the consequences of biodiversity changes at larger spatial scales, in particular biotic homogenization, that is, reduced species turnover across space (ß diversity), remain poorly known. Using data from 39 grassland biodiversity experiments, we examine the effects of ß diversity on the stability of simulated landscapes while controlling for potentially confounding biotic and abiotic factors. Our results show that higher ß diversity generates more asynchronous dynamics among local communities and thereby contributes to the stability of ecosystem productivity at larger spatial scales. We further quantify the relative contributions of α and ß diversity to ecosystem stability and find a relatively stronger effect of α diversity, possibly due to the limited spatial scale of our experiments. The stabilizing effects of both α and ß diversity lead to a positive diversity-stability relationship at the landscape scale. Our findings demonstrate the destabilizing effect of biotic homogenization and suggest that biodiversity should be conserved at multiple spatial scales to maintain the stability of ecosystem functions and services.

Strong impact of management regimes on rhizome biomass across Central European temperate grasslands.

Grassland ecosystems account for approximately 40% of terrestrial biomes globally. These communities are characterized by a large allocation to belowground biomass, often exceeding its aboveground counterpart. However, this biomass investment cannot be entirely attributed to the acquisitive function of roots. Grassland plants also allocate to non-acquisitive, stem-derived, belowground organs, such as rhizomes. These organs are responsible for the key plant functions of space occupancy, resprouting after damage, and seasonal rest. However, biomass investment to rhizomes has rarely been studied. Here we gathered community-level aboveground and rhizome biomass data for 52 temperate grasslands in Czech Republic (Central Europe), differing in management intensity. We found that rhizome biomass scaled linearly with aboveground biomass, and more intensive management disproportionally (negatively) affected rhizome biomass. This finding may have important implications for the persistence of temperate grassland plants and their provision of ecosystem services (e.g., soil carbon sequestration, soil stabilization) in relation to changing environments.

Synchrony matters more than species richness in plant community stability at a global scale.

The stability of ecological communities is critical for the stable provisioning of ecosystem services, such as food and forage production, carbon sequestration, and soil fertility. Greater biodiversity is expected to enhance stability across years by decreasing synchrony among species, but the drivers of stability in nature remain poorly resolved. Our analysis of time series from 79 datasets across the world showed that stability was associated more strongly with the degree of synchrony among dominant species than with species richness. The relatively weak influence of species richness is consistent with theory predicting that the effect of richness on stability weakens when synchrony is higher than expected under random fluctuations, which was the case in most communities. Land management, nutrient addition, and climate change treatments had relatively weak and varying effects on stability, modifying how species richness, synchrony, and stability interact. Our results demonstrate the prevalence of biotic drivers on ecosystem stability, with the potential for environmental drivers to alter the intricate relationship among richness, synchrony, and stability.

Experimental assessment of biotic and abiotic filters driving community composition.

Species occurrence in a site can be limited by both the abiotic environment and biotic interactions. These two factors operate in concert, but their relative importance is often unclear. By experimentally introducing seeds or plants into competition-free gaps or into the intact vegetation, we can disentangle the biotic and abiotic effects on plant establishment. We established a seed-sowing/transplant experiment in three different meadows. Species were introduced, as seeds and pregrown transplants, into competition-free gaps and the intact vegetation. They included 12 resident plants from the locality and 18 species typical for different habitats. Last two years, gaps were overgrown with vegetation from surrounding plants and we observed the competitive exclusion of our focal plants. We compared plant survival with the expected occurrence in target locality (Beals index). Many of the species with habitat preferences different from our localities were able to successfully establish from seeds and grow in the focal habitat if competition was removed. They included species typical for much drier conditions. These species were thus not limited by the abiotic conditions, but by competition. Pregrown transplants were less sensitive to competition, when compared to seedlings germinated from seeds. Beals index significantly predicted both species success in gaps and the ability to withstand competition. Survival in a community is dependent on the adaptation to both the abiotic environment and biotic interactions. Statistically significant correlation coefficients of the ratio of seedling survival in vegetation and gaps with Beals index suggest the importance of biotic interactions as a determinant of plant community composition. To disentangle the importance of abiotic and biotic effect on plant establishment, it is important to distinguish between species pool as a set of species typically found in given community type (determined by Beals index) and a set of species for which the abiotic conditions are suitable.

Trait probability density (TPD): measuring functional diversity across scales based on TPD with R.

Functional diversity (FD) has the potential to address many ecological questions, from impacts of global change on biodiversity to ecological restoration. There are several methods estimating the different components of FD. However, most of these methods can only be computed at limited spatial scales and cannot account for intraspecific trait variability (ITV), despite its significant contribution to FD. Trait probability density (TPD) functions (which explicitly account for ITV) reflect the probabilistic nature of niches. By doing so, the TPD approach reconciles existing methods for estimating FD within a unifying framework, allowing FD to be partitioned seamlessly across multiple scales (from individuals to species, and from local to global scales), and accounting for ITV. We present methods to estimate TPD functions at different spatial scales and probabilistic implementations of several FD concepts, including the primary components of FD (functional richness, evenness, and divergence), functional redundancy, functional rarity, and solutions to decompose beta FD into nested and unique components. The TPD framework has the potential to unify and expand analyses of functional ecology across scales, capturing the probabilistic and multidimensional nature of FD. The R package TPD (https://CRAN.R-project.org/package=TPD) will allow users to achieve more comparative results across regions and case studies.

The legacy of initial sowing after 20 years of ex-arable land colonisation.

Priority effects provide an advantage to early establishing species and are thought to significantly affect the course of succession. We conducted a 20-year long experiment sowing high- and low-diversity mixtures in an ex-arable field. We ask how long the effect of sowing persists and which sown species affect the course of succession. The experiment was established in the Czech Republic in five replicate blocks, each containing three random 10 × 10 m plots with three treatments: natural colonisation, sowing low- and high-diversity seed mixtures. The species cover was annually estimated in 12 permanent 1 m2 quadrates within each plot. To identify the effects of sowing, we used an innovative method analysing the data separately for each year using Redundancy analysis (RDA) with identity of sown species as explanatory variables. In the first year, the effect of sowing was small; the peak of explained variability occurred between third and fifth year. The legacy of sowing was detectable in the natural colonisers for 18 years and in the sown species for the whole 20-year period. For some species, the difference between the plots where they were and were not sown remained significant for the whole 20-year period (e.g. Lathyrus pratensis) although the plots were adjacent and the area was mown with the same machine. Other ones (e.g. Trisetum flavescens) colonised all the plots evenly. The long-lasting effect of the initial sowing confirms contingency of successional pathway on the propagule pressure in the time of start of succession due to the priority effects.

Functional differences stabilize beetle communities by weakening interspecific temporal synchrony.

The temporal stability of communities is essential for the maintenance of ecosystem functioning across trophic levels. The stabilizing effect of biodiversity is, among other factors, modulated by the level of synchrony in population fluctuations among the species in the community. What drives community synchrony, however, remains largely unclear. Community synchrony can be affected by external drivers such as disturbances, but also by the properties of the community. Species with different ecological strategies should fluctuate less synchronously than more similar species; thus, an increase in diversity of ecological strategies should decrease synchrony, and increase the stability of the community. Here, using an exceptionally large data set of ground beetle trappings in Dutch heathlands (~370,000 individuals in 19 plots, each sampled between 9 and 36 yr), we assess the drivers of community stability and synchrony, and their relationship with disturbance, species richness, and functional diversity (FD). We found no effect of disturbance (fire and topsoil removal) on community stability or synchrony, probably because of unpredictable patterns of increase or decrease of the populations. Community synchrony was overall positive, giving more support for independent and positive correlation between species than for compensatory dynamics. Synchrony decreased with increasing FD, but not with species richness. Supporting this, we found that the more species pairs differ in their traits, the less synchronously their populations fluctuate, where 74% of all pairs showed no significant correlation. Significant positive synchrony (19% of species pairs) was concentrated among pairs with low trait dissimilarity, and the 7% of pairs with significant negative temporal correlation showed no relation with pairwise functional dissimilarity. The stabilizing effect of FD via decreased synchrony supports largely untested theoretical expectations that an increased diversity of functional strategies in a community will have a stabilizing effect on community abundance. We hypothesize that because competition is low in this community, the stabilizing effect of FD reflects interspecific variation in responses to environmental fluctuations rather than competition.

Tasty rewards for ants: differences in elaiosome and seed metabolite profiles are consistent across species and reflect taxonomic relatedness.

Diaspores of myrmecochorous plants consist of a seed (or fruit) and an attached appendage (elaiosome) which attracts ants. The elaiosome is a food resource for ants, whereas the seed is an energy source for subsequent germination and plant establishment. Although myrmecochory occurs in many phylogenetically unrelated lineages, multiple phylogenetic lineages display similar variation in elaiosome and seed metabolite composition due to convergent evolution. We focused on four families (Amaryllidaceae, Boraginaceae, Papaveraceae and Poaceae) each represented by two species from different genera. Diaspores of three populations per species were sampled and concentrations of 60 metabolites from five groups (amino acids, fatty acids, organic acids, polyols and sugars) were determined for both elaiosomes and seeds. Variability in metabolite composition was decomposed by hierarchical ANOVA and variation partitioning using redundancy analysis (reflecting both species nested within families, crossed with seed vs. elaiosome). Differences in the metabolite composition of elaiosomes and seeds were consistent across multiple phylogenetic origins (with more pronounced differences at the level of individual metabolites than at the level of metabolite groups) and supported the idea of convergent evolution under strong selection pressure. Elaiosomes contained higher amounts of easily digestible metabolites (especially amino acids) than seeds. Fatty acids were not more concentrated in elaiosomes, which contradicts the literal translation of "elaiosome" (= oil body). The differentiation of metabolite composition closely reflected taxonomic relatedness, particularly at the family level. Differences among populations within species were small, so the metabolite composition can thus be considered as a trait with relatively low intraspecific variability.

Impact of herbivory and competition on lake ecosystem structure: underwater experimental manipulation.

Two basic ecological relationships, herbivory and competition, distinctively influence terrestrial ecosystem characteristics, such as plant cover, species richness and species composition. We conducted a cage experiment under natural conditions in an aquatic ecosystem to test the impacts of two treatments combined in a factorial manner: (i) a pulse treatment - removal of dominant competitors among primary producers (macroalgae Chara sp. and Vaucheria sp.), and (ii) a press treatment - preventing herbivore (fish, crayfish) access to caged plots. The plots were sampled once before the treatments were established and four more times within two years. Both treatments had a significantly positive impact on macrophyte cover and species richness and changed the macrophyte species composition. The effect of the macroalgae removal was immediate with the highest species richness occurrence during the first post-treatment monitoring, but the positive effect vanished with time. In contrast, preventing herbivore access had a gradual but long-lasting effect and reached a more steady-state over time. Two of the most common species showed contrasting responses, the palatable Potamogeton pectinatus was most supported by caging, while the distasteful Myriophyllum spicatum preferred open plots. Our findings may be applicable during the revitalisation of aquatic ecosystems that aims to increase macrophyte biodiversity.

Stabilizing effects in temporal fluctuations: management, traits, and species richness in high-diversity communities.

The loss of biodiversity is thought to have adverse effects on multiple ecosystem functions, including the decline of community stability. Decreased diversity reduces the strength of the portfolio effect, a mechanism stabilizing community temporal fluctuations. Community stability is also expected to decrease with greater variability in individual species populations and with synchrony of their fluctuations. In semi-natural meadows, eutrophication is one of the most important drivers of diversity decline; it is expected to increase species fluctuations and synchrony among them, all effects leading to lower community stability. With a 16-year time series of biomass data from a temperate species-rich meadow with fertilization and removal of the dominant species, we assessed population biomass temporal (co)variation under different management types and competition intensity, and in relation to species functional traits and to species diversity. Whereas the effect of dominant removal was relatively small (with a tendency toward lower stability), fertilization markedly decreased community stability (i.e., increased coefficient of variation in the total biomass) and species diversity. On average, the fluctuations of individual populations were mutually independent, with a slight tendency toward synchrony in unfertilized plots, and a tendency toward compensatory dynamics in fertilized plots and no effects of removal. The marked decrease of synchrony with fertilization, contrary to the majority of the results reported previously, follows the predictions of increased compensatory dynamics with increased asymmetric competition for light in a more productive environment. Synchrony increased also with species functional similarity stressing the importance of shared ecological strategies in driving similar species responses to weather fluctuations. As expected, the decrease of temporal stability of total biomass was mainly related to the decrease of species richness, with its effect remaining significant also after accounting for fertilization. The weakening of the portfolio effect with species richness decline is a crucial driver of community destabilization. However, the positive effect of species richness on temporal stability of total biomass was not due to increased compensatory dynamics, since synchrony increased with species richness. This shows that the negative effect of eutrophication on community stability does not operate through increasing synchrony, but through the reduction of diversity.

Testing the environmental filtering concept in global drylands.

1. The environmental filtering hypothesis predicts that the abiotic environment selects species with similar trait values within communities. Testing this hypothesis along multiple - and interacting - gradients of climate and soil variables constitutes a great opportunity to better understand and predict the responses of plant communities to ongoing environmental changes. 2. Based on two key plant traits, maximum plant height and specific leaf area (SLA), we assessed the filtering effects of climate (mean annual temperature and precipitation, precipitation seasonality), soil characteristics (soil pH, sand content and total phosphorus) and all potential interactions on the functional structure and diversity of 124 dryland communities spread over the globe. The functional structure and diversity of dryland communities were quantified using the mean, variance, skewness and kurtosis of plant trait distributions. 3. The models accurately explained the observed variations in functional trait diversity across the 124 communities studied. All models included interactions among factors, i.e. climate - climate (9% of explanatory power), climate - soil (24% of explanatory power) and soil - soil interactions (5% of explanatory power). Precipitation seasonality was the main driver of maximum plant height, and interacted with mean annual temperature and precipitation. Soil pH mediated the filtering effects of climate and sand content on SLA. Our results also revealed that communities characterized by a low variance can also exhibit low kurtosis values, indicating that functionally contrasting species can co-occur even in communities with narrow ranges of trait values. 4. Synthesis We identified the particular set of conditions under which the environmental filtering hypothesis operates in drylands worldwide. Our findings also indicate that species with functionally contrasting strategies can still co-occur locally, even under prevailing environmental filtering. Interactions between sources of environmental stress should be therefore included in global trait-based studies, as this will help to further anticipate where the effects of environmental filtering will impact plant trait diversity under climate change.

Applying the dark diversity concept to nature conservation.

Linking diversity to biological processes is central for developing informed and effective conservation decisions. Unfortunately, observable patterns provide only a proportion of the information necessary for fully understanding the mechanisms and processes acting on a particular population or community. We suggest conservation managers use the often overlooked information relative to species absences and pay particular attention to dark diversity (i.e., a set of species that are absent from a site but that could disperse to and establish there, in other words, the absent portion of a habitat-specific species pool). Together with existing ecological metrics, concepts, and conservation tools, dark diversity can be used to complement and further develop conservation prioritization and management decisions through an understanding of biodiversity relativized by its potential (i.e., its species pool). Furthermore, through a detailed understanding of the population, community, and functional dark diversity, the restoration potential of degraded habitats can be more rigorously assessed and so to the likelihood of successful species invasions. We suggest the application of the dark diversity concept is currently an underappreciated source of information that is valuable for conservation applications ranging from macroscale conservation prioritization to more locally scaled restoration ecology and the management of invasive species.

Measuring size and composition of species pools: a comparison of dark diversity estimates.

Ecological theory and biodiversity conservation have traditionally relied on the number of species recorded at a site, but it is agreed that site richness represents only a portion of the species that can inhabit particular ecological conditions, that is, the habitat-specific species pool. Knowledge of the species pool at different sites enables meaningful comparisons of biodiversity and provides insights into processes of biodiversity formation. Empirical studies, however, are limited due to conceptual and methodological difficulties in determining both the size and composition of the absent part of species pools, the so-called dark diversity. We used >50,000 vegetation plots from 18 types of habitats throughout the Czech Republic, most of which served as a training dataset and 1083 as a subset of test sites. These data were used to compare predicted results from three quantitative methods with those of previously published expert estimates based on species habitat preferences: (1) species co-occurrence based on Beals' smoothing approach; (2) species ecological requirements, with envelopes around community mean Ellenberg values; and (3) species distribution models, using species environmental niches modeled by Biomod software. Dark diversity estimates were compared at both plot and habitat levels, and each method was applied in different configurations. While there were some differences in the results obtained by different methods, particularly at the plot level, there was a clear convergence, especially at the habitat level. The better convergence at the habitat level reflects less variation in local environmental conditions, whereas variation at the plot level is an effect of each particular method. The co-occurrence agreed closest the expert estimate, followed by the method based on species ecological requirements. We conclude that several analytical methods can estimate species pools of given habitats. However, the strengths and weaknesses of different methods need attention, especially when dark diversity is estimated at the plot level.