Richness and taxonomic distinctness of cave invertebrates from the northeastern state of Goiás , central Brazil : a vulnerable and singular area

Abstract The karst area of the northeastern state of Goiás comprises two main municipalities: São Domingos and Posse. São Domingos is inside the limits of a Full Protection Conservation Unit known as Parque Estadual de Terra Ronca (PETeR), where a high number of caves occurs, some of them surpassing 10 km in length. Despite their protection by law, uncontrolled tourism has been threatening the integrity of the unique and fragile cave systems of São Domingos. On the other hand, the caves of Posse are much less visited, with its subterranean fauna poorly unknown and are strongly threatened by the urban areas and mining activities in the vicinity. We conducted six systematic surveys of invertebrates in 12 caves, seven of which are located in São Domingos and five in Posse, between 2010 and 2012. Immediately outside the caves, we sampled several microhabitats for comparison. We estimated richness, abundance, and taxonomic distinctness of the communities. We recorded 1,941 individuals of 344 morphospecies. Caves from São Domingos showed a higher species richness and were more taxonomic distinct than caves from Posse. Most morphospecies were considered troglophiles (188). We also found one trogloxene and one troglobite, as well as nine troglomorphic taxa that possibly are troglobitic as well. Both regions have markedly singular caves regarding its subterranean fauna, with high values of taxonomic distinctness. However, the richer caves were not necessarily the most taxonomic distinct. Conservation measures are especially necessary in the region of Posse, where caves are not legally protected and are within an area of significant urbanization. Cave entrances of Posse are near deforested vicinities, in some cases with domestic wastes and cement plants nearby. Subterranean Biology 29: 1–33 (2019)


Introduction
The subterranean fauna consists of organisms able to survive in peculiar conditions of the environment, as the permanent darkness, the high relative humidity of the air and stable temperatures at the deepest zones (Barr 1968).These inherent features may act as an environmental filter (see Fernandes et al. 2016 to opposite view) in such a way that only part of the epigean taxa is capable to colonize and establish hypogean populations.So, the organisms found inside caves can be sorted in three ecologicalevolutionary categories, considering its degree of specialization and dependence of surface environments: trogloxenes, troglophiles and troglobites.
Because of its unique conditions and the high degree of endemism, subterranean environments are fragile and sensitive to environmental changes, especially those regarding anthropic activities (Culver 1986, Gibert and Deharveng 2002, Trajano and Bichuette 2006).Among the main human interference in Brazilian caves, we may stress mining, construction of hydroelectric dams, deforestation for agriculture and uncontrolled tourism (Ferreira et al. 2014, Gallão andBichuette 2018).
Brazil has about 17,000 registered caves (CECAV 2018).Some heterogeneity exists in the knowledge about the subterranean fauna regarding different Brazilian karst and non-karst areas.Some are well known, as the Vale do Ribeira (southeastern) (e.g., Trajano 1991, Bichuette and Trajano 2003, Rodrigues et al. 2014) and Chapada Diamantina (eastern-northeastern) (Gregorin and Mendes 1999, Bockmann and Castro 2010, Gallão and Bichuette 2015).Others, however, have its knowledge limited to preliminary data about species richness and abundance, which was obtained on a few sampling occasions (e.g.Altamira, northern) (Trajano and Moreira 2001).Besides this, only a few approaches covered long-term samplings, with replicas enough to contemplate more than one annual cycle (e.g.Trajano 1991, Gallão andBichuette 2015, Resende and Bichuette 2016, Zepon andBichuette 2017).Most often in tropical caves, the seasonal oscillation is of great importance in the dynamics of the subterranean environments (Trajano 2000).
Some studies have shown that phylogenetic diversity, a measure of species relatedness considering the phylogenetic relationship among species, are more sensitive to detecting responses of communities to environmental changes (Cianciaruso et al. 2009).This is because traditional diversity indexes have limited prediction about ecosystems functioning.According to Faith (1992), the emphasis on conservation regards preserving as much of this hierarchical variation as possible, no matter what the taxonomic identity involved.In this sense, a phylogenetic diversity index has been shown more efficient to proposals of conservation strategies than traditional indexes (Cianciaruso et al. 2009) and was used also for conservation purposes comparing taxonomic distinctness with α indexes (Gallão and Bichuette 2015).
In the present study, we sampled subterranean invertebrates at the karst area of the northeastern Goiás state, central Brazil, encompassing the municipalities of São Domingos and Posse.We estimated species richness and abundance by each cave and by each region, as well as by the karst area.Finally, we compared the fauna of these caves regarding geographical position and taxonomic distinctness of the communities.We expected that phylogenetic component of diversity would indicate those most singular caves in the region better than traditional indexes of diversity.Our purpose was to improve the knowledge about components of biological diversity in the region and, through a better knowledge of the ecological-evolutionary processes underlying these communities, provide best arguments for better conservation decisions.

Study Area
The karst area at northeastern Goiás represents one of the regional expressions of Bambuí geomorphological Unit, the largest set of limestone in Brazil, comprising approximately 105,200 km 2 (Auler et al. 2001).Surficial rivers belonging to the Paranã basin (Alto Tocantins) permeate the limestone after draining an extensive sandstone area, forming large cave systems (Karmann andSetúbal 1984, Auler andFarrant 1996).
Part of São Domingos region is inside the limits of a Conservation Unit, the Parque Estadual de Terra Ronca (PETeR, Figure 1), which was created in 1989, but still with many land ownership conflicts and inspection problems, resulting in timber harvesting and trampling by cattle near the headwaters.Additionally, it is aggravating the fact that the headwaters of the main rivers that flow through the cave systems are outside the limits of the Conservation Unit (Gallão and Bichuette 2012).São Domingos possess complex systems of surficial and subterranean drainage, with high potential of organic matter carriage and accumulation of debris in some caves.Based upon this, these caves present high richness of subterranean fauna, whether terrestrial or aquatic (Pinto-da-Rocha 1995, Rheims and Pelegatti-Franco 2003, Majer et al. 2003, Simões et al. 2013).Seven troglobitic species of fishes have been recorded so far, configuring a high diversity for this group (Bichuette 2003, Bichuette andTrajano 2003).
Posse, which represents the southernmost part of the same limestone outcrop, is 200 km from PETeR (Figure 1).In there, the systems and subterranean drainage are not as well developed as they are in São Domingos.Not all caves from Posse in this study are mapped yet, or their maps are still being produced; the Russão cave, which is currently being mapped, have an estimated development of, at least, 4 km (E.C.Igual pers.comm.).
Both regions are inside the limits of Cerrado Domain (Ab'Saber 1977), with a dry season between March and September, which may extend until October (Nimer 1979).Rainfall pattern is marked by floods in the rainiest periods (October to March), which bring large amounts of organic matter inside the caves.
We visited the study area on six occasions, three during the raining season (April 2010, April 2011 and February 2012) and three during the dry season (October 2011, June and October 2012).Caves varied with respect to the number of visits: two visits, one during the dry season and other during the raining season in Lapa do Bezerra, São Bernardo and Terra Ronca I, two during the raining season in São Mateus, two during the dry season in Terra Ronca II, one during the dry season in Pau Pombo and Revolucionários, and one during the raining season in Bombas, Doralino, Nova Esperança and Russão.Lapa Angélica was the only cave monitored on all occasions.
We sampled terrestrial substrates and streams mainly by active search in all potential microhabitats, including piles of leaf litter, trunks, guano deposits, under rocks, among others, where specimens were collected with tweezers and brushes and immediately euthanized and preserved in 70% alcohol.In two caves, Lapa Angélica and Terra Ronca II, we also used 0.25 m² quadrats (Bichuette et al. 2015).For comparative purposes, i.e., to define ecological-evolutionary categories of taxa, we also sampled in surface habitats near the caves (ca 50 to 100 m from entrance).The collections were performed during night and daytime by active search in leaf litter piles, tree trunks, and fallen trunks, and by transects (10 m of length).We also removed soil by excavation method for more accurate evaluation at the laboratory, and we used Winkler extractor to collect fauna associated with leaf litter.
We identified collected specimens to the least inclusive taxonomic level possible (Operational Taxonomic Units / OTUs) using specific literature as a guide (Borror and DeLong 1969, Adis 2002, Brescovit et al. 2002, Zeppelini and Bellini 2004, Rafael et al. 2012).Part of the material was sent to taxonomic specialists for confirmation and more accurate identification.In cases when identification to the species level could not be obtained, we used higher-level hierarchies and classified specimens into morphotypes and, thus, they are reliable taxonomic categories.Immature individuals that could not be clearly attributed to adults already collected were excluded from all analyses.

Data analysis
All taxa we found were listed for each cave and study area (Appendix 1).Additionally, we defined ecological-evolutionary categories of the better-known taxa following the proposal of Trajano (2012) comparing with the epigean fauna, personal knowledge, and literature data.We also generated histograms of species richness and abundance towards a better understanding of the faunistic composition by region as well as by the Goiás karst area.
We determined the taxonomic distinctness of each cave using the index of phylogenetic diversity known as taxonomic distinctness (TD, Δ*) (Warwick and Clarke 1995), using the package Vegan (Oksanen et al. 2018) in the software R (version 3.4.0)(R Core Team 2017).This index compares the phylogenetic relationship in a community considering each locality (in our case, the caves).Thus, caves with closely phylogenetic species show lower TD in relation to caves with species phylogenetic distinct.Higher TD values show singularity of an environment.Index of TD is calculated as the number of estimated nodes to connect two different species in a community, while the randomization estimates the confidence interval.This index is applicable to our data because it does not depend on the number of samples or abundance.Then, we did a simple linear regression in the software R (version 3.4.0)(R Core Team 2017) correlating richness and TD values for each area, in order to verify if these data are related.

Results
We collected a total of 1,941 individuals of 344 morphospecies belonging to 128 families, 37 orders and ten classes (Appendix 1) in both regions: 287 morphospecies and 1,554 individuals were from São Domingos and 122 morphospecies and 387 individuals were from Posse.Whether we consider São Domingos and Posse singly or combined as a larger karst area, insects and arachnids were the richest and more abundant taxa (Figure 2-4).

Discussion
Our data show high species richness for both the São Domingos and Posse karst areas, which has been also observed for other Brazilian karst areas, such as Chapada Diamantina with c.a. 160 morphospecies (Gallão and Bichuette 2015) and Presidente Olegário with 382 morphospecies (Zepon and Bichuette 2017).Many species that we found during this study were often reported from other limestone caves whether in the same karst area or in other Brazilian karst areas (Trajano 1987, Pinto-da-Rocha 1995, Zepon and Bichuette 2017).As expected for Brazilian caves, most species we found are troglophiles (Trajano and Bichuette 2010).
In São Domingos karst area, Lapa Angélica cave was the richest and the most abundant.Although we sampled this cave on more occasions, its high richness is likely due to the high input of nutrients brought by the extense rivers crossing the cave systems and, consequently, to the high amount of dissolved organic carbon in subterranean terrestrial substrates (62.2 mgC.L -1 ); the same was observed for Terra Ronca II cave (111.12 mgC.L -1 ) (Paula 2018), which, in addition, has skylights along its length through which more organic matter may enter the system (Bichuette et al. 2017).In relation to Pau Pombo cave, the low richness and abundance values may be explained by the limited habitat we searched compared to other caves, as well as this is a typicall dry cave.
In Posse karst area, the higher species richness and abundance we found inside the cave Revolucionários is also attributable to the drainage traversing its length, as well as the high humidity of the terrestrial substrates and the high amount of trophic resources, composed mainly of leaf litter, frugivorous and hematophagous bat guano (Gnaspini-Neto 1992, Poulson andLavoie 2000).Low diversity and abundance at Nova Esperança can be attributed to low amount of organic matter found inside the cave, mainly leaf litter and guano (ME Bichuette pers.obs).It is a small cave, crossed by a river and with few terrestrial habitats available for fauna, most of which contain rocky or clay substrates.There are several skylights at the ceiling and its epigean environs are impacted by the presence of cattle and pastures.
São Bernardo (São Domingos region) and Revolucionários (Posse region) were the most singular caves in Goiás karst area.Gallão and Bichuette (2015) also observed a high singularity for two caves from Bahia state, northeastern Brazil (Gruna Parede Vermelha and Morro de Alvo caves).Not necessarily the richest caves had higher TD.In other words, the richest caves hadn't the highest singularities in their faunistic compositions.For instance, Lapa Angélica, the richest cave of São Domingos, had a value of TD lower than the estimated mean for the region.This is related to the great number of morphospecies shared with other caves in the region.Our results reinforce that is inadequate to consider only the observed local diversity as the sole criterion for relevance attribution of an area, as this index by itself is not effective to compare different environments, regions, nor taxa (Pärtel et al. 2011).Numerical values of alpha diversity do not reflect the obvious singularities of the subterranean habitats, which is the main justification for its conservation (Trajano et al. 2012).
Lapa Angélica is also one of the most visited caves from PETeR and, on some occasions, visitors traverse its entire length.There has been no effective control of the number and frequency of tourist groups in the region since 2000 (ME Bichuette pers.obs.).We noted that there is only a few sparsely guano spots along the cave, probably reduced as a consequence of anthropic impacts (ground trampling and noise pollution, which drives off the bats).Disturbance diminishes the environmental heterogeneity and, consequently, reduces the offer of microhabitats and resources to the local community, as observed in Lapa Angélica in relation to bat guano along years (M.E.Bichuette pers.obs.).Environmental impacts (contamination) also cause reduction of taxonomic distinctness in marine species communities, while richness remained constant (Warwick and Clarke 1998).
We observed that some of the other richest caves which do not have high taxonomic distinctness, possibly because of anthropic impacts in the region.Taxonomic distinctness in the region may still be related to the trophic diversity of the community, in such a way that when there is a reduction in the number of guilds, TD decays (Clarke and Warwick 1999).The type of habitat affects trophic diversity.Consequently, more impacted areas are also more subject to a decrease in taxonomic distinctness.
Terra Ronca II cave, even with the second higher species richness, presented the lowest TD.The stretch of the cave we sampled has basically the same substratum type, which is predominantly composed of banks of sand and pebbles deposited at riverside.Despite its high species richness, the species are not taxonomically very distinct from each other (e.g., several morphospecies are from the beetle families Carabidae and Staphylinidae) when compared to other subterranean communities of the same region.In Posse, the richest cave (Revolucionários) also had the highest TD.Its high faunistic singularity is related to the presence of species and genera belonging to different families.Nova Esperança presented several common taxa and, therefore, low faunistic singularity.
If we excluded the troglophilic taxa and considered only troglobitic terrestrial invertebrates, species diversity recorded in northeastern Goiás would be considered poor when compared to other Brazilian regions, as has been demonstrated in this and in previous studies (Trajano andBichuette 2010, Trajano et al. 2016).This poor troglobitic diversity can be related to the fact that this area comprises a relatively stable paleoclimatic zone (Barr 1973, Moore 1964).Under this model of climate fluctuations, areas where drastic climate fluctuations occurred in the past had greater troglobitic diversification, as can be found in the subterranean fauna in the northern Hemisphere (Barr 1968, Peck 1980).The most specialized Brazilian troglobites are found in caves of semiarid regions of Bahia state, as Chapada Diamantina and Campo Formoso (Trajano 1995).
As we corroborated in our study, a high degree of singularity is usually present in caves, even among those, which are geographically near other caves and inserted in the same rocky massif.To preserve as many singular caves as possible is urgent, because the ideal of environmental compensation is not applicable to these unique environments.
Even the caves from São Domingos are inside a legally protected area (PETeR), the tourist flow is intense and, several times, there is no adequate inspection.Thus, intense visitation may be affecting the subterranean communities we studied.Besides, part of the headwaters of rivers that crosses caves of São Domingos is outside the State Park limits.There, extensive cattle farming and monocultures are causing silting and pollution of rivers, bringing noxious materials to the caves and impacting the community (Simões et al. 2013).In February 2013, intense sedimentation occurred at the headwater of the São Vicente River (São Domingos), as a consequence of intensive agriculture upstream of the spring.In view of the above, it is difficult to preserve natural areas, even when legal reserve areas and parks are delimited.In Posse, the surroundings outside the caves are deforested and replaced by pastures and urban areas, while the rivers are being discharged from domestic sewage.Worryingly, Brazilian environmental laws do not legally protect the caves suffering these impacts.
In conclusion, caves of São Domingos and Posse karst areas exhibited a high taxonomic distinctness for terrestrial fauna, influenced by the high proportion of troglophilic, accidental and undetermined taxa.Our data show the relevance to considerer not only troglobitic taxa.Besides non-troglobionts, both areas showed a high diversity of troglobitic fish (Bichuette and Trajano 2003) and shelters highly important for bats (Bichuette et al. 2018).Anthropogenic impacts cause reduction of environmental heterogeneity and trophic diversity, and, consequently, diminishes faunistic singularity.Besides, these karst areas have high faunistic diversity when compared with other Brazilian regions.Therefore, protective measures should be taken, since the region is not totally preserved, even in the São Domingos area, where the surroundings of the headspring of rivers that enter on the caves (responsible for carrying food resources and increasing heterogeneity of habitats), are still outside the protection limits of the Park.

Figure 1 .
Figure 1.Map showing sampled caves from the São Domingos karst area (blue circles) within Terra Ronca State Park (PETeR, green area) and from the Posse karst area (red diamonds).FD, Federal District.

Figure 2 .
Figure 2. A Richness and B abundance of subterranean terrestrial invertebrate (by Class) from the São Domingos and Posse karst areas, state of Goiás, and from both regions combined.