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.

cave, completeness, isopods, species pool

Subterranean biodiversity patterns are marked by taxonomic and functional disharmony (sensu Cardoso 2012) and by high species turnover (Gibert and Deharveng 2002) when compared to the surface fauna in the same region. On the one side, taxonomic and functional disharmony occurs when species or functional groups are missing from the subterranean environment, because some taxa may be favoured to the detriment of other taxa during the occupation of caves (Cardoso 2012). On the other side, high species turnover reflects the smaller ranges of obligate cave fauna (Barr and Holsinger 1985; Gibert and Deharveng 2002). The processes underlying these patterns are generally attributed to regional factors, such as high habitat fragmentation and long term stability (Gibert and Deharveng 2002), and to local influences, such as resource and habitat availability, competition, and predation (Culver 1981; Christman and Culver 2001; Culver 2012; Resende and Bichuette 2016). Biodiversity, thus, bears an imprint of both influences (He et al. 2005) in such a way that species do not occur in all caves or habitat patches where their occurrences are expected (Culver and Pipan 2009; Culver 2012; Resende and Bichuette 2016). One way to understand the impact of these patterns on subterranean communities is by looking for those species whose presence was expected but are absent instead.

Observable patterns cannot always be completely explained by ecological mechanisms, because they reflect only part of the bigger picture (Lewis et al. 2017). Some species occur in the region, tolerate the conditions of the environment, but are not present in the focal area. The knowledge of this kind of absences can complement the understanding of ecological processes and increase the effectiveness of conservation strategies (Lewis et al. 2017). This concept is known as dark diversity (Pärtel et al. 2011). Understanding which species are in the dark diversity, why they are absent, and the consequences to community processes sheds light on several ecological processes still not completely understood or predicted by observed patterns of species distribution. Using dark diversity, one may better understand species distributions (Riibak et al. 2015), predict the restoration potential of degraded habitats, the likelihood of successful species invasions (Lewis et al 2017) or even future distributions of threatened species under a climate change scenario (Tang et al. 2019). Possibilities are innumerable. For example, in subterranean communities we may question the implications of dark diversity in terms of community saturation and how this information can be applied to cave conservation.

Underlying the concept of dark diversity is the idea of how much of the species pool is realised within a community (Pärtel et al. 2013). With such information complementing other methods, ecologists may adapt informed and effective strategies for conservation and restoration (Lewis et al. 2017). Calculating the proportion of species absences (dark diversity) in relation to presences (observed richness) enables one to understand which part of the observed richness is natural variation in the regional pool size and which part is dark diversity (Pärtel et al. 2013; Pärtel 2014). An index known as community completeness (Pärtel et al. 2013) allows us to estimate this proportion and it is calculated as the logistic expression between observed richness and dark diversity (log_e(observed richness / dark diversity)). Since species richness and dark diversity covary, community completeness shows no relationship with latitude, allowing the comparison of different communities and regions (Ronk et al. 2015).

Even though this concept of biodiversity has been encouraged and discussed since its definition by Pärtel et al. (2011), only a few studies effectively applied dark diversity to enhance the prediction power of ecological processes (e.g. Pärtel 2014; Riibak et al. 2015; Ronk et al. 2015; Tang et al. 2019) and to encourage its importance in supporting well informed management decisions (Lewis et al. 2017). Some controversy about the determination of species pool also has been raised (Mokany and Paini 2011), but methods already applied to the determination of suitable habitats, using Beals probability index based on co-occurrences (Beals 1984; Münsbergová and Herben 2004) or in Ellenberg indicator values proved applicable to dark diversity as well (Lewis et al. 2016). Despite this central role in understanding biodiversity and its importance in conservation and management, the concept of dark diversity has been applied only to plant communities, probably because of the availability of plant databases at several spatial scales. No study has applied dark diversity to subterranean communities to date. In the midst of this debate, we propose that the application of dark diversity to subterranean communities is feasible and interesting for conservation purposes.

We used an assemblage of terrestrial isopods (Oniscidea) as a model taxon for evaluating the dark diversity of caves. Isopods are a very diversified and widespread group not only in superficial but also in subterranean environments, particularly in caves, where they benefit from the milder conditions, high humidity, and the great variety of favourable substrates (Taiti 2003; Campos-Filho et al. 2014). Phylogenetically related taxa were used to set the species pool and to appropriately assess dark diversity, because only a few species of isopods co-occur inside caves (Fernandes et al. 2016). We expected subterranean communities to have high dark diversity and low completeness, because of their typically high beta diversity (Gibert and Deharveng 2000). Therefore, we aimed to compare how much of the habitat-specific regional isopod species pool was found within each local cave community. In addition, we advocate for the application of the dark diversity concept in ecological studies and monitoring of subterranean ecosystems to help guide conservation policies.

By comparing the Beals Index distribution at both occupied and unoccupied habitats, we set the threshold value at 0.559, which was the median of the occupied habitats. Above this Beals Index value, an unoccupied habitat had 50% of probability of being occupied, representing suitable habitats for the presence of isopods already present in the species pool.

In the five caves we sampled, we found six species of isopods. On average, only two species inhabited each cave (mean ± s.d. = 2.2 ± 1.30), with the richest cave, Lapa do Angélica, having four species. Most species were in the dark diversity (3.2 ± 1.3), even when a given species was distributed in other nearby caves. Only Lapa do Angélica had positive value of completeness, with more species present than absent; the others had negative values, with more species in the dark diversity (Table 1).

Cerrado is well preserved and is continually present outside all caves of São Domingos, allowing dispersal of the fauna via surface environments. Subterranean dispersal may also occur via the innumerable interconnected cracks and crevices typical of the karst, because the limestone outcrops in São Domingos are continuous (Bichuette and Trajano 2003). Despite this, isopods that were widespread on surface and caves nearby did not occur in all the caves where they potentially could.

Even if each cave is unique in its shape and resource inputs, almost all caves had dark diversity equal or higher than observed richness, with low completeness. From a conservation perspective, the caves with higher completeness may be a source of migratory individuals to other areas in the vicinity, which may suffer impacts and local extinctions (Lewis et al. 2017). Therefore, Lapa do Angélica, the only cave with positive value of completeness, has the higher regional relevance, acting as a potential repository of diversity to nearby caves.

Lapa do Angélica is an extensive cave with great input of organic matter during the rainy season. In its 14 km of horizontal projection, the cave is crossed by a river with almost 7 km of subterranean routes with stretches of rapids and several rainfalls resulting in high amount of organic matter being imported and transported from adjacent communities (Auler et al. 2001; Bichuette and Trajano 2003). These conditions provide high diversity of microhabitats and food sources for the fauna, probably enabling the coexistence of the four species of isopods found there and minimising the number of species in the dark diversity. Lapa do Bezerra, on the other hand, was the least diverse in isopods and had the lowest completeness, despite its more than 8 km of horizontal projection traversed by almost 4 km of a river with high transport capacity accumulating great amounts of organic matter inside the cave (Bichuette and Trajano 2003). Lapa do Bezerra and Lapa do Angélica are part of the same cave system (Angélica-Bezerra cave system), possibly connected to each other and both with similar altimetric gradients (around 120 meters) (Guyot et al. 1996). It is possible that their proximity influenced the expected occurrences based on shared fauna and resulted in the lowest completeness of Lapa do Bezerra, just as the isopods in the dark diversity may be part of the fauna not discovered yet (hidden diversity sensu Pärtel 2014). Both Lapa do Bezerra and Lapa do São Bernardo caves have river beds with narrow margins, with narrower deposits of sediments and organic matter when compared to Lapa do Angélica (Auler et al. 2001). During the rainy season, organic matter is washed away from these microhabitats and the fauna can hide in inaccessible passages of the cave. On the one hand, the community can be saturated at lower richness, because of the lower input of organic matter. On the other, some species that are present may not have been sampled yet, because of their behavior facing the conditions of the environment. Consequently, both the hidden diversity and the completeness can be influenced by the shape of the caves.

Setting conservation priorities is essential, because funding sources are finite and several societal interests are involved (Díaz et al. 2011). Coupled with already existing conservation tools, dark diversity and completeness can be a helpful tool to aid and direct conservation efforts. Through an accurate assessing of completeness, researchers and managers can evaluate and monitor restoration of degraded habitats, avoid the risk of species invasion, and direct efforts to preserve a valuable sample of regional species pool (Lewis et al. 2017). This can be especially important in areas with caves and its irreplaceable and often vulnerable specialised fauna. Understanding the local species pools and how the species are distributed in caves within a region can help to adapt conservation efforts to preserve a representative sample of the subterranean fauna, from which at least part of lost species can be recouped. Our study showed that the dark diversity approach using patterns of co-occurrence among phylogenetically related taxa is adaptable to subterranean communities and may be the kickoff for both applied and theoretical studies aiming the better comprehension of ecological processes underlying patterns of subterranean biodiversity.

We would like to thank JE Gallão, PP Rizzato, LB Simões, DM Von-Schimonsky, and T Zepon, for collections; to Ramiro H dos Santos for guiding us in the field; to JE Gallão and to DM Von-Schimonsky for suggestions in the manuscript; to A. Gambarini for the images in Figure 2; to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), for the scholarship granted to the first author; to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), for the scholarships granted to the second and third authors (MAB: 305912/2013-5; MEB: 303715/2011-1 and 57413/2014-0); to the Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp, grant 2010/08459-4) and Programa de Pós-Graduação em Ecologia e Recursos Naturais, for the financial support; to the Secretaria de Meio Ambiente e Recursos Hídricos (SEMARH n°063/2012) and Parque Estadual de Terra Ronca (PETER) for the authorisation for scientific research in the conservation unit; to the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBIO, 20165-1), for the sampling permit. This study was financed in part by CAPES, Finance Code 001. We are also grateful to Dr. Stefano Mammola and Dr. Matthew L. Niemiller for their valuable criticisms and suggestions which improved our paper.