Dark Diversity Research Articles

Effects of plant characteristics and environmental factors on the dark diversity in a broadleaved Korean pine forest

Effects of plant characteristics and environmental factors on the dark diversity in a broadleaved Korean pine forest

Determinants of species assemblages of insect pests in alpine forest ecosystems of western China

BackgroundInsect pests are a significant threat to natural resources and social development. Modeling species assemblages of insect pests can predict spatiotemporal pest dynamics. However, research gaps remain regarding the mechanism for determining species assemblages of insect pests in alpine forest ecosystems. Here, we explored these determinants using a field investigation conducted for insect pests in a region of the Qinghai-Tibet Plateau. We assessed the species assemblages of insect pests in alpine forest ecosystems based on species co-occurrence patterns and species diversity (i.e., observed diversity, dark diversity, community completeness, and species pool). A probabilistic model was used to test for statistically significant pairwise patterns of species co-occurrence using the presence-absence matrix of pest species based on species interactions. We used ordinary least squares regression modeling to explore relationships between abiotic factors (i.e., climate factors and human influence) and species diversity.ResultsPositive pest species interactions and many association links can occur widely across different investigation sites and parts of plant hosts in alpine forest ecosystems. We detected high dark diversity and low community completeness of insect pests in alpine forest ecosystems. High temperature and precipitation could promote pest species diversity, particularly dark diversity and species pools. Human influence could drive high levels of pest species diversity and lead to dark diversity and species pools. Community completeness could be an effective indicator for insect pest risk assessment.ConclusionsOur study provides new evidence for the determinants of insect pest species assemblages in alpine forest ecosystems from the perspectives of pest species interactions and abiotic factors. The findings of our study could reveal the mechanism for shaping species assemblages and support the prevention and control of insect pests in alpine forest ecosystems.

Determining macrophyte species richness and dark diversity sources – A novel approach to improve the biodiversity estimation based on species traits

Biodiversity measures deliver valuable ecological information by reflecting a range of ecosystem processes. However, the accuracy of environmental assessment based on species patterns may often be affected by insufficient survey details. The comprehensive evaluation of plant taxa richness in rivers requires an extensive sampling effort. The use of Hill numbers and Chao estimators improves species diversity assessment based on a feasible number of samples. The aim of this work was to identify macrophyte groups, associated with various species traits, which are rich in species, as well as those whose detection is particularly difficult as it requires an exceptional sampling effort (sources of dark diversity). Analyses were performed with the use of Hill numbers and Chao estimators. It was shown that the field identification of all estimated macrophytes is particularly difficult for low trophy indicators and generally submerged plants, as well as for small-leaved species. A field survey encompassing the full (expected) macrophyte diversity encountered within a river is easiest to perform in the case of free-floating plants and large-leaved macrophytes, as well as for species with high trophic tolerance. The study proved that ecological assessment of rivers based on a small number of sampling units may lead to incorrect diversity estimates. Conversely, the estimation of diversity patterns at the level of the Shannon and Simpson indices does not require extensive sampling, and the extrapolation approach is not needed. The effectiveness of diversity assessment in fluvial ecosystems can be increased by extrapolation of gray diversity which can be considered in planning of monitoring programs. Moreover even estimated dark diversity bight be already efficient to identify ecological pattern and when comparing biodiversity across regions and ecosystems.

From taxonomic to functional dark diversity: Exploring the causes of potential biodiversity and its implications for conservation

Abstract The conservation of natural habitats requires an accurate assessment of both their degradation and their potential for restoration. Among the tools available, dark diversity is promising, since it estimates all the species that could be present in a community due to favourable environmental conditions but are currently lacking. However, current experimental approaches measure dark diversity mainly from species co‐occurrences, which rely partly on neutral processes, and also suffer from several problems concerning the inclusion of rare species. Here, we propose a novel method to estimate dark diversity, which is based on more deterministic co‐occurrence and also strongly limits the problems of rarity: the co‐occurrence of species' functional features. We adapted the Beals' co‐occurrence index using functional groups, and we estimated functional dark diversity based on co‐occurrence of functional groups. To illustrate the interests of our approach, we then addressed as an example a major issue of restoration ecology: how does passive rewilding (i.e. spontaneous restoration of woodlands) impact the ecological integrity of recovered communities? We compared post‐agricultural woodlands with ancient forests, in terms of taxonomic and functional dark diversity of vascular plants and spiders. For both taxa, the patterns of dark diversity in recent woodlands and ancient forests changed depending on whether we used the taxonomic or functional approach. This was due to the detection of functional features even if they were hosted by rare species, and to the consideration of ecological redundancy between species in functional groups. Notably, recent woodlands harboured plant communities with a similar amount of taxonomic dark diversity compared to ancient forests, but with less functional dark diversity. Also, the composition of functional dark diversity clearly differed between forest types. Synthesis and applications. Functional dark diversity brings novel perspectives for ecological diagnostic and restoration. In particular, it provides a more mechanistic understanding of the constraints which limit the re‐assembly of natural communities during restoration practices (e.g. dispersal limitation). The functional approach usefully complements the taxonomical one to identify habitat conservation and restoration interests, especially for highly degraded sites where the detection of missing groups via species is biased because of their rarity.

Dark diversity of flea assemblages of small mammalian hosts: effects of environment, host traits and host phylogeny

An assemblage of species in a locality comprises two components, namely (i) species that are present (realised diversity) and (ii) species from the regional pool that may potentially inhabit this locality due to suitable ecological conditions, but that are absent (dark diversity). We investigated factors affecting the dark diversity of component communities of fleas parasitic on small mammals in the northern Palearctic at two scales. First, we considered the dark diversity of flea assemblages of the same host (for 13 host species) across regions and tested for the effects of environmental factors and the number of available host species on the dark diversity of within-region flea assemblages. Second, we considered the dark diversity of fleas across host species within a region (for 20 regions) and asked whether within-host dark diversity is associated with host phylogeny and/or traits. We found that the dark diversity of flea assemblages harboured by small mammals varied substantially (i) within the same host species across space (in 12 of 13 host species) and (ii) between host species within a region (in eight of 20 regions). The size of the dark diversity of flea assemblages of the same host across regions was generally affected by environmental factors (mainly by the amount of green vegetation), whereas the size of the dark diversity of flea assemblages of a host species within a region was affected by host traits (mainly by the degree of host sociality and the structure of its shelter and, to a lesser degree, by its geographic range size) but was not associated with host phylogenetic affinities. We conclude that application of the dark diversity concept to parasite communities across space or hosts allows a better understanding of the factors affecting the species richness and composition of these communities.

Rarity facets of biodiversity: Integrating Zeta diversity and Dark diversity to understand the nature of commonness and rarity.

Measuring commonness and rarity is pivotal to ecology and conservation. Zeta diversity, the average number of species shared by multiple sets of assemblages, and Dark diversity, the number of species that could occur in an assemblage but are missing, have been recently proposed to capture two aspects of the commonness‐rarity spectrum. Despite a shared focus on commonness and rarity, thus far, Zeta and Dark diversities have been assessed separately. Here, we review these two frameworks and suggest their integration into a unified paradigm of the “rarity facets of biodiversity.” This can be achieved by partitioning Alpha and Beta diversities into five components (the Zeta, Eta, Theta, Iota, and Kappa rarity facets) defined based on the commonness and rarity of species. Each facet is assessed in traditional and multiassemblage fashions to bridge conceptual differences between Dark diversity and Zeta diversity. We discuss applications of the rarity facets including comparing the taxonomic, functional, and phylogenetic diversity of rare and common species, or measuring species' prevalence in different facets as a metric of species rarity. The rarity facets integrate two emergent paradigms in biodiversity science to better understand the ecology of commonness and rarity, an important endeavor in a time of widespread changes in biodiversity across the Earth.

Integrating dark diversity and functional traits to enhance nature conservation of epiphytic lichens: a case study from Northern Italy

Mountains provide a timely opportunity to examine the potential effects of climate change on biodiversity. However, nature conservation in mountain areas have mostly focused on the observed part of biodiversity, not revealing the suitable but absent species—dark diversity. Dark diversity allows calculating the community completeness, indicating whether sites should be restored (low completeness) or conserved (high completeness). Functional traits can be added, showing what groups should be focused on. Here we assessed changes in taxonomic and functional observed and dark diversity of epiphytic lichens along elevational transects in Northern Italy spruce forests. Eight transects (900–1900 m) were selected, resulting in 48 plots and 240 trees, in which lichens were sampled using four quadrats per tree (10 × 50 cm). Dark diversity was estimated based on species co-occurrence (Beals index). We considered functional traits related to growth form, photobiont type and reproductive strategy. Linear and Dirichlet regressions were used to examine changes in taxonomic metrics and functional traits along gradient. Our results showed that all taxonomic metrics increased with elevation and functional traits of lichens differed between observed and dark diversity. At low elevations, due to low completeness and harsh conditions, both restoration and conservation activities are needed, focusing on crustose species. Towards high elevations, conservation is more important to prevent species pool losses, focusing on macrolichens, lichens with Trentepohlia and sexual reproduction. Finally, dark diversity and functional traits provide a novel tool to enhance nature conservation, indicating particular threatened groups, creating windows of opportunities to protect species from both local and regional extinctions.

Relationships between macro-fungal dark diversity and habitat parameters using LiDAR

Despite the important role of fungi for ecosystems, relatively little is known about the factors underlying the dynamics of their diversity. Moreover, studies do not typically consider their dark diversity: the species absent from an otherwise suitable site. Here, we examined potential drivers of local fungal dark diversity in temperate woodland and open habitats using LiDAR and in-situ field measurements, combined with a systematically collected and geographically comprehensive macro-fungi and plant data set. For the first time, we also estimated species pools of fungi by considering both plant and fungi co-occurrences. The most important LiDAR variables for explaining fungal dark diversity were amplitude and echo ratio, which represent vegetation structure. These results suggest that the local fungal dark diversity is highest in production forests like plantations and lowest in more open forests and in open habitats with little woody vegetation. Plant species richness was the strongest explanatory factor overall and negatively correlated with local fungal dark diversity. Soil fertility showed a positive relationship with dark diversity in open habitats. These findings indicate that the local dark diversity of macro-fungi is highest in areas with a relatively high human impact (typically areas with low plant species richness and high soil fertility). Overall, this study brings novel insights into local macro-fungi dark diversity patterns, suggesting that a multitude of drivers related to both soil and vegetation act simultaneously to determine fungal dark diversity.

Estimating probabilistic site‐specific species pools and dark diversity from co‐occurrence data

AbstractAimThe species pool specific for a site includes all species from the region that are theoretically able to live in the site's particular ecological conditions. The absent portion of the site‐specific species pool forms the site’s dark diversity, which is unobservable and can only be estimated. Most existing methods to designate dark diversity act in a binary fashion. Here, we argue that the species pool is more suitably defined as a fuzzy set, present a method to estimate probabilistic species pools using pairwise co‐occurrence data with a hypergeometric distribution, and compare it with established methods (Beals index and favourability correction).InnovationWe compare the different aspects of the method’s performance using simulations based on individual agents in which the suitability for each species in each site is known. Further, we assessed the methods in two real datasets with nested sampling designs. We provide the R package ‘DarkDiv’ (https://cran.r‐project.org/web/packages/DarkDiv/index.html) that implements all the compared methods for estimations of probabilistic dark diversity and species pools.Main conclusionsBeals method is extremely sensitive to species frequency, and predicts species’ suitability to local conditions less accurately than the other considered methods. The favourability transformation corrected this relationship, but still predicted extremely low probabilities for species with very little information. The hypergeometric method outperformed the Beals and favourability methods in all considered aspects in the simulations and displayed better characteristics in the real datasets. The hypergeometric method is currently the best option to estimate probabilistic dark diversity and species pool composition based on pairwise species co‐occurrence data.

Community completeness as a measure of restoration success: multiple-study comparisons across ecosystems and ecological groups

When restoring habitat for biodiversity, the most effective outcome will be achieved by restoration projects which target several organism groups or ecosystem types. Such integrated approaches require direct comparisons among different ecological communities while evaluating success of restoration. The Community Completeness Index (CCI) is a recently developed metric that allows such comparisons by accounting for both present and absent but otherwise suitable taxa. We empirically evaluated the applicability of CCI for assessing the outcome of ecological restoration. We analyzed how species richness and the completeness of ecological communities recover after restoration, for different ecological groups and ecosystem types, and how it develops over time after restoration. Analyses were performed on 18 datasets with per site presence-absence data from Northern Europe. Each dataset represented one of the three habitat types (mire, forest, grassland) and different ecological groups (plants, flying insects, epigeic invertebrates). Datasets contained pristine, degraded and restored sites. We calculated the dark diversity and subsequently CCI based on species co-occurrences. Our multiple-study analyses revealed that CCI of grassland plant communities increased faster after restoration than invertebrate communities or plant communities in forests and mires. In addition, flying insect communities demonstrated significantly highest CCI in pristine mires. Some results were significant only for richness but not for CCI indicating species pool effect. Finally, completeness and species richness of restored communities increased with time since restoration. As such, our study demonstrated that CCI is a useful tool in evaluating restoration success across different organism groups and ecosystem types.

Dark Diversity in Restorations: What’s missing?

Dark Diversity in Restorations: What’s missing?

Functional traits determine why species belong to the dark diversity in a dry grassland fragmented landscape

A challenge for nature conservation is to know why many species are absent from suitable habitats and whether they might be able to disperse and to establish. Here, we used 272 dry grassland patches within a fragmented landscape to investigate the role of local abiotic conditions and dispersal filtering in determining the likelihood of vascular plants to belong to the dark diversity (i.e. absent portion of the species pool). First, we quantified the species (SD), functional (FD) and phylogenetic (PD) diversity of both observed and dark communities. Second, we determined the roles of abiotic, present‐day and historical landscape configuration variables in shaping their patterns. Third, we evaluated the importance of each variable in determining their species. Environmental filtering was assessed as effects of local abiotic conditions and dispersal filtering as the effects of present‐day and historical landscape configuration. Dispersal filtering was also estimated by comparing dispersal traits of observed and dark diversity. Finally, we assessed community completeness to determine how much of the species pool was realized within a local community. We found higher SD in the observed compared to the dark communities, but PD did not differ. Contrary to expectations, dark communities resembled higher FD compared to the observed communities. Species with low dispersal capacity, low competitive abilities and high stress‐tolerance were more often absent. Observed and dark diversities were mostly affected by local abiotic variables. In the observed communities, present‐day landscape configuration variables affected SD while historical landscape configuration variables explained FD and PD. In the dark communities, we found the opposite pattern. Completeness was affected by present‐day and historical patch size. Our results explain why dry grassland species may belong to the dark diversity and highlight the importance of local abiotic and dispersal traits of the species to conserve dry grasslands in changing landscapes.

Drivers of plant community completeness differ at regional and landscape scales

Although many grassland species may exist within agricultural landscapes, some are still absent from individual grassland patches. Understanding which processes limit plant biodiversity within these landscapes is important, and may be better understood if we consider local diversity relative to its species pool size (community completeness). Here we test how habitat availability, dispersal limitation, grassland age, and local environmental conditions (productivity, fine scale heterogeneity) affect the completeness of grassland plant communities at two spatial scales in an agricultural region in Southeast Estonia, Northern Europe. We additionally test how species dispersal and persistence traits (seed production, potential dispersal distance, and clonal ability) depend on the landscape and local environmental factors.We collected vascular plant data from 31 grassland patches and used species co-occurrences to estimate which species from the surrounding region were suitable, but absent for each grassland site (dark diversity). We partitioned dark diversity for each site into species that were found within the surrounding 10 ha area (landscape scale) or species found only at other visited grassland sites (regional scale), and used these estimates to calculate completeness for each site at the landscape and regional scale. Environmental data (various soil characteristics, light availability, plant biomass) was collected to characterize productivity and fine scale heterogeneity of the sites.We found that community completeness was limited by seed production ability, dispersal distances, and grassland availability at the regional scale, whereas local environmental conditions (productivity) limited completeness at the landscape scale. Species dispersing long distances were more often present in areas historically bordered by more grasslands, whereas potential dispersal limitation was reduced in older grasslands with heterogeneous and sparse vegetation.We conclude that dispersal limitation, habitat availability, and local environmental conditions all determine completeness of grassland patches; however, their relative importance is scale dependent. Nevertheless, the presence of good dispersers in isolated grasslands is likely a disappearing legacy of historical landscapes. To support high taxonomic and functional diversity in agricultural landscapes, it is therefore crucial to increase the area and connectivity of grassland patches and maintain their moderate management by mowing or grazing.

Dark diversity reveals importance of biotic resources and competition for plant diversity across habitats.

Species richness is the most commonly used metric to quantify biodiversity. However, examining dark diversity, the group of missing species which can potentially inhabit a site, can provide a more thorough understanding of the processes influencing observed biodiversity and help evaluate the restoration potential of local habitats. So far, dark diversity has mainly been studied for specific habitats or large‐scale landscapes, while less attention has been given to variation across broad environmental gradients or as a result of local conditions and biotic interactions. In this study, we investigate the importance of local environmental conditions in determining dark diversity and observed richness in plant communities across broad environmental gradients. Using the ecospace concept, we investigate how these biodiversity measures relate to abiotic gradients (defined as position), availability of biotic resources (defined as expansion), spatiotemporal extent of habitats (defined as continuity), and species interactions through competition. Position variables were important for both observed diversity and dark diversity, some with quadratic relationships, for example, plant richness showing a unimodal response to soil fertility corresponding to the intermediate productivity hypothesis. Interspecific competition represented by community mean Grime C had a negative effect on plant species richness. Besides position‐related variables, organic carbon was the most important variable for dark diversity, indicating that in late‐succession habitats such as forests and shrubs, dark diversity is generally low. The importance of highly competitive species indicates that intermediate disturbance, such as grazing, may facilitate higher species richness and lower dark diversity.

Deriving site‐specific species pools from large databases

Defining the species pool of a community is crucial for many types of ecological analyses, providing a foundation to metacommunity, null modelling or dark diversity frameworks. It is a challenge to derive the species pool empirically from large and heterogeneous databases. Here, we propose a method to define a site‐specific species pool (SSSP), i.e. the probabilistic set of species that may co‐occur with the species of a target community. Using large databases with geo‐referenced records that comprise full plant community surveys, our approach characterizes each site by its own species pool without requiring a pre‐defined habitat classification. We calculate the probabilities of each species in the database to occur in the target community using Beals’ index of sociological favourability, and then build sample‐based rarefaction curves from neighbouring records with similar species composition to estimate the asymptotic species pool size. A corresponding number of species is then selected among the species having the highest occurrence probability, thus defining both size and composition of the species pool. We tested the robustness of this approach by comparing SSSPs obtained with different spatial extents and dissimilarity thresholds, fitting different models to the rarefaction curves, and comparing the results obtained when using Beals co‐occurrence probabilities or presence/absence data. As an example application, we calculated the SSSPs for all calcareous grassland records in the German Vegetation Reference Database, and show how our method could be used to 1) produce grain‐dependent estimations of species richness across plots, 2) derive scalable maps of species richness and 3) define the full list of species composing the SSSP of each target site. By deriving the species pool exclusively from community characteristics, the SSSP framework presented here provides a robust approach to bridge biodiversity estimations across spatial scales.

Temporal lags in observed and dark diversity in the Anthropocene.

Understanding biodiversity changes in the Anthropocene (e.g. due to climate and land-use change) is an urgent ecological issue. This important task is challenging because global change effects and species responses are dependent on the spatial scales considered. Furthermore, responses are often not immediate. However, both scale and time delay issues can be tackled when, at each study site, we consider dynamics in both observed and dark diversity. Dark diversity includes those species in the region that can potentially establish and thrive in the local sites' conditions but are currently locally absent. Effectively, dark diversity connects biodiversity at the study site to the regional scales and defines the site-specific species pool (observed and dark diversity together). With dark diversity, it is possible to decompose species gains and losses into two space-related components: one associated with local dynamics (species moving from observed to dark diversity and vice versa) and another related to gains and losses of site-specific species pool (species moving to and from the pool after regional immigration, regional extinction or change in local ecological conditions). Extinction debt and immigration credit are useful to understand dynamics in observed diversity, but delays might happen in species pool changes as well. In this opinion piece we suggest that considering both observed and dark diversity and their temporal dynamics provides a deeper understanding of biodiversity changes. Considering both observed and dark diversity creates opportunities to improve conservation by allowing to identify species that are likely to go regionally extinct as well as foreseeing which of the species that newly arrive to the region are more likely to colonize local sites. Finally, by considering temporal lags and species gains and losses in observed and dark diversity, we combine phenomena at both spatial and temporal scales, providing a novel tool to examine biodiversity change in the Anthropocene.

Dark diversity in the dark: a new approach to subterranean conservation

When trying to predict biodiversity patterns, species absences in a community can be as informative as species presences. The concept of dark diversity considers geographical and ecological filters to set an expected species pool and to compare it with the observed species pool, through an index known as community completeness. Completeness shows no relationship with latitude, allowing the comparison of different communities and regions concerning community saturation. Here we propose the use of these methods to a better understanding of subterranean biodiversity patterns. We applied patterns of co-occurrence among phylogenetically related species to set the theoretical species pool and then compared it with the observed richness, using isopods as model taxon. Except for one cave, dark diversity was equal or higher than observed richness. Even though completeness was low in most cases, those caves with higher completeness represent a valuable sample of regional subterranean species pool and may act as a repository of diversity. Our study showed that the dark diversity approach is adaptable to studies of subterranean communities and may be coupled with other conservation tools towards more effective management decisions.

DarkDivNet – A global research collaboration to explore the dark diversity of plant communities

AbstractDarkDivNet is a global research collaboration which explores dark diversity — the set of species that are absent from a site despite being suitable under the site conditions and present in the region. Participants of the network survey vascular plant diversity both at local (10 m × 10 m) and regional scales (radius 10 km) using a standardized approach. They also measure simple plant traits and collect soil samples. Observed and dark diversity together form the site‐specific species pool, and the ratio of observed diversity and dark diversity describes community completeness. We shall explore how observed and dark diversity, site‐specific species pool and community completeness vary across natural and anthropogenic gradients. We link plant diversity measures to the information obtained from environmental DNA: soil biota and plant taxa that occurred at the site before. We will refine existing dark diversity methods and use large vegetation databases to infer species habitat suitability. We expand the dark diversity concept from a purely taxonomy‐based approach to include the functional and phylogenetic aspects of diversity. DarkDivNet currently includes 161 planned sampling areas globally, but new participants are welcome. The main vegetation sampling period is scheduled until September 2020, with the first research papers being produced after that.

Come to the dark side! The role of functional traits in shaping dark diversity patterns of south‐eastern European hoverflies

1. Dark diversity represents the set of species that can potentially inhabit a given area under particular ecological conditions, but are currently ‘missing’ from a site. This concept allows characterisation of the mechanisms determining why species are sometimes absent from an area that seems ecologically suitable for them.2. The aim of this study was to determine the dark diversity of hoverflies in south‐eastern Europe and to discuss the role of different functional traits that might increase the likelihood of species contributing to dark diversity. Based on expert opinion, the Syrph the Net database and known occurrences of species, the study estimated species pools, and observed and dark diversities within each of 11 defined vegetation types for 564 hoverfly species registered in south‐eastern Europe. To detect the most important functional traits contributing to species being in dark diversity across different vegetation types, a random forest algorithm and respective statistics for variable importance were used.3. The highest dark diversity was found for southwest Balkan sub‐Mediterranean mixed oak forest type, whereas the lowest was in Mediterranean mixed forest type. Three larval feeding modes (saproxylic, and phytophagous on bulbs or roots) were found to be most important for determining the probability of a species contributing to hoverfly dark diversity, based on univariate correlations and random forest analysis.4. This study shows that studying dark diversity might provide important insights into what drives community assembly in south‐eastern European hoverflies, especially its missing components, and contributes to more precise conservation prioritisation of both hoverfly species and their habitats.

A novel method to predict dark diversity using unconstrained ordination analysis

AbstractQuestionsSpecies pools are the product of complex ecological and evolutionary mechanisms, operating over a range of spatial scales. Here, we focus on species absent from local sites but with the potential to establish within communities — known as dark diversity. Methods for estimating dark diversity are still being developed and need to be compared, as well as tested for the type, and amount, of reference data needed to calibrate these methods.LocationSouth Bohemia (48°58′ N, 14°28′ E) and Železné Hory (49°52′ N, 15°34′ E), Czech Republic.MethodWe compared a widely accepted algorithm to estimate species pools (Beals smoothing index, based on species co‐occurrence) against a novel method based on an unconstrained ordination (UNO). Following previous work, we used spatially nested sampling for target plots, with the dark diversity estimates computed from smaller plots validated against additional species present in larger plots, and a reference dataset (Czech National Phytosociological Database of >30,000 plots as global reference data). We determined which method provides the best estimate of dark diversity with an index termed the “Success Rate Index”.ResultsWhen using the whole reference dataset (national scale), both UNO and Beals provided comparable predictions of dark diversity that were better than null expectations based on species frequency. However, when predicting from regionally restricted spatial scales, UNO performed significantly better than Beals. UNO also tended to detect less common species better than Beals. The success rate of combining UNO and Beals slightly outperformed the results obtained from the single methods, but only with the largest reference dataset.ConclusionsThe UNO method provides a consistently reliable estimate of dark diversity, particularly when the reference dataset is size‐limited. For future calculations, we urge caution regarding the choice of dark diversity methods with respect to the reference data available, and how different methods handle species of high, and low, occurrence frequency.