Taxonomic Distinctness of the subterranean fauna from Peruaçu Caves National Park, state of Minas Gerais, eastern Brazil

Limiting factors of subterranean environments, high relative air humidity and, especially, permanent darkness, represent ecological filters for organisms and biodiversity patterns of surface and subterranean communities display wide disparities. Subterranean diversity and singularity are, thus, better expressed when the common presence of rare and endemic species are considered. Our study aimed to describe the diversity of the cave fauna from 14 caves from Peruaçu Caves National Park (PCNP), eastern Brazil. We analyzed the regional diversity using the index that includes the average taxonomic distinction (TD Δ +, AvTD). We recorded 1,674 individuals belonging to 10 Classes and 237 morphotypes, 11 troglobitic and two troglomorphic and possible troglobites. Greatest species abundance (N=330) and richness (s=76) were recorded at Lapa do Cipó cave, followed by Gruta Olhos d’Água (N=330, s=71), which shows the highest TD value (Δ + = 90.18) in relation to the others, including the richest Lapa do Cipó (Δ + = 85.24), consequence of the several taxonomic units with large number of species. The below-expected values of TD (Δ + = 87.70) may indicate anthropic impacts on these communities. Our results demonstrate that Gruta Olhos d’ Água and Lapa do Cipó caves are the most important sites for the occurrence of endemic and troglobitic species and may be part of a complex system that should be considered for a more efficient conservation planning.


Introduction
Subterranean or hypogean environments have striking features which represent environmental filters, because selective regimen contrasts sharply with those of surface environments. Permanent darkness at deeper zones is the main difference (Hoenen 2005, Hervant et al. 2000, Poulson & White 1969, leading to the dependence of allochthonous energy inputs because photoautotrophic organisms do not thrive there (Poulson & Lavoie 2000). Environmental stability is other noteworthy aspect of subterranean ecosystems. Unlike surface communities, little variation occurs in parameters such temperature and air humidity, due to the isolation caused by the surrounding rock (Juberthie 2000, Culver & Pipan 2009). Such contrasts between subterranean and superficial environments are related to processes of colonisation and reproductive isolation of subterranean species (Trajano 2012).
Subterranean communities are formed by populations which present distinct ecological-evolutionary relationships with the environment and, consequently, may be classified at three categories (Racovitza 1907, Trajano 2012, according to its degree of specialisation to the subterranean realm: trogloxenes are those who need to leave the cave periodically in order to complete its life cycle because subterranean resources are insufficient (Racovitza 1907). Troglophiles are those capable of completing its life cycle inside as well as outside subterranean environments. They represent the majority of the taxa found at subterranean communities (Racovitza 1907). Some species only live at subterranean environments (Racovitza 1907), not being able to colonise surface environments anymore, because they evolved isolated at subterranean environments and, during this period, accumulated specialisations that hampers survival or reproduction in surface environments. They are called troglobites (Racovitza, 1907). This condition is recognised by the presence of autapomorphies acquired throughout evolutionary process, known as troglomorphisms (Holsinger & Culver 1988, Holsinger 1988, Trajano & Bichuette 2010. Even though the term troglomorphism (sensu Christiansen 1962) had been originally proposed for morphological characteristics, it has been applying to behavioural and physiological characteristics as well (Barr 1968). This classification has been extended and discussed by Trajano and Cobolli (2012) to include source-population concept and, thus, population characteristics has been included for recognizing the troglobitic status of a given species.
Diversity patterns of subterranean and superficial communities show significant disparities. Even though richness of subterranean communities is much lower when compared to superficial communities, it can be considered high on a global scale (Gibert & Deharveng 2002). In addition, when lineages that colonised both habitats at the same geographical area are compared, subterranean communities have much higher proportion of endemic species than superficial communities (Gibert & Deharveng 2002). Recognition of such patterns is essential to support conservation proposals (Trajano 2010).
Knowledge about Brazilian subterranean fauna has been broadened at the last two decades. The first faunistic lists were produced for the karst area of Ribeira Valley (Trajano & Gnaspini 1991), states of São Paulo and Paraná (Trajano & Bichuette 2010). Later, other regions had their diversity assessed, including karst areas at the states of Minas Gerais, Bahia, Mato Grosso do Sul, Goiás and Rio Grande do Norte (Gnaspini & Trajano 1994, Pinto-da-Rocha 1993, Trajano & Bichuette 2010, Ferreira et al. 2010, Gallão & Bichuette 2018. Some are important because they have high species richness, presence of phylogenetic indicators or high genetic diversity. (Trajano et al. 2016). High genetic diversity is based on the degree of specialisation of individual species, with accumulation of autapomorphies, that may evolve into a troglobitic species, while phylogenetic indicators are related to the presence of biogeographical relicts (Trajano et al. 2016). Exclusively subterranean populations are generally fragile and represent one of the main arguments in favour of the conservation of caves and its systems (Trajano 2010, Trajano et al. 2016, Gallão & Bichuette 2018. For this reason, to understand the evolutionary processes and patterns that occur in underground environments, faunal studies are needed to identify high diversity spots and to propose actions and political for conservation. (Trajano et al. 2016).
We investigated the biodiversity of caves located at Peruaçu Caves National Park (PCNP), northern state of Minas Gerais, taking into account not only the number of species, but its identity as well. The region has 140 caves registered so far, of which, Gruta Olhos d' Água cave is the largest one, with approximately 9,100 m of conduits and passageways, and harbors the highest number of troglobites (Trajano et al. 2016, Gallão & Bichuette 2018. For this, two main questions were considered: 1) Do the PCNP caves have high phylogenetic diversity? 2) Does Gruta Olhos d 'Água cave present higher diversity compared to the other caves of the PCNP?

Study Area
We conducted the study herein presented in 14 caves located at Peruaçu Caves National Park (PCNP), municipalities of Itacarambi and Januária, northern of Minas Gerais state ( Figure 1). The region has outcrops composed by carbonate rocks from Bambuí Group, more precisely laminated limestone and dolomites from Januária-Itacarambi Formation (Piló & Kohler 1991). Inside the limits of PCNP, Peruaçu river traverses about 17 Km of its valley surrounded by enormous rocky walls, channels and dolines, forming the Peruaçu river, left tributary of the upper-middle São Francisco River.
Peruaçu Valley, where PCNP is located, occurs in a transition zone between Cerrado and Caatinga morphoclimatic Domains (Ab'Saber 1977). Climate covering Peruaçu, according to Köppen-Geiger, is tropical humid with dry winters ("Aw") (Peel et al. 2007), characterised by dry winters, between March and October, and rainy summers, between April and November, with annual average temperature of 24°C and average annual rainfall of 800 mm (INMET 2010).  We sampled potential microhabitats by active search and then the collected specimens were euthanized and preserved in 50% and 70% alcohol. We then identified morphotypes at the least inclusive taxonomic level we could reach using specialised literature (Borror et al., 1989;Adis, 2002;Costa et al., 2006). In addition, we consulted specialists in order to refine identification: Bolfarini,

Data analysis
We used Taxonomic Distinctness index (TD) (Δ+, AvTD) for diversity analysis, defined as the mean distance between two randomly chosen species, traced by means of a Linnean or phylogenetic classification of all the species in the data-set (Warwick & Clarke 1998, Clarke & Warwick 2001).
Differently of a traditional diversity index, the Taxonomic Distinctness TD (Δ+, AvTD) take into account the abundance of rare and common species by giving them different weights (Warwick & Clarke 1998, Cianciaruso et al. 2009). Besides considering the relative contribution of each species at a given community by using the number of species and the distribution of individuals, TD also includes the taxonomic value of each species.
Taxonomic distinctness is, thus, estimated by the expected number of nodes between any two individuals of different species randomly drawn from the full species set. The biggest number of nodes or links among them, the higher will be the distinctness of a given species (Warwick & Clarke 1998, Cianciaruso et al. 2009). Also, Taxonomic Distinctness index is a robust measure, when compared to traditional indexes of diversity, because it is independent of sampling effort (Warwick & Clarke 1998). We used PRIMER 7 statistical package to calculate TD (Δ+, AvTD) (Clarke & Gorley 2015) using the data-set.
The PCNP has 11 troglobitic species described (Table 1)    Development and extension of aphotic zones were the most contrasting physical parameters when comparing the caves (Table 2). Gruta Olhos d' Água is the longest cave with an extensive aphotic zone in the region, Gruta do Janelão presents large galleries and an extensive twilight zone with several openings to the epigean environment.
The other caves show small extensions, some without aphotic zones or perennial drainages, such as Lapa do Sonho, Toca do Pedrinho and Gruta Mina d' Água. A general characterization of each cave is presented at Table 2.

Discussion
Subterranean fauna of PCNP is remarkable. Our results corroborate the hypothesis that Gruta Olhos d'Água cave is a spot of subterranean biodiversity (Trajano et al. 2016) probably due the long period of environmental stability, as evidenced by its high troglobitic species richness, at least 13 so far (Perez-González et al. 2017, Gallão & Bichuette 2018. In addition, we identified another important locality of endemic and troglobitic species, the Lapa do Cipó cave (Table 4).
Besides the great number of taxa, we also recorded a significant number of troglobites at Peruaçu region. For Brazil, the highest number of troglobites is recorded at Areias System, located at the Alto Ribeira karst area, state of São Paulo, accounting for 20 species described so far, followed by Alambari de Cima cave, at the same region, with 10 troglobites (Trajano & Bichuette 2010, Trajano et al. 2016. Other spot of high subterranean diversity (Gallão & Bichuette 2015, Trajano et al. 2016 has been identified at Chapada Diamantina region, at the state of Bahia, with 23 troglobites recorded at 11 caves, 13 of them from a single cave. The analysis of taxonomic distinction (Δ+, AvTD) takes into account that the more phylogenetically distinguishes species in a given community, the greater the diversity (Cianciaruso et al. 2009). However, when taxonomic relationships are taken into account, high TD values (Δ+, AvTD) indicate high diversity preserved spots (Clarke & Warwick 2001), as we observed for Gruta Olhos d 'Água (s = 71, Δ+ = 90.18) ( Figure 5). On the other hand, despite presenting higher richness, Lapa do Cipó presented a lower than expected TD (s = 76, Δ+ = 85.24), indicating possible degradation and / or the existence of some genera with many species (Figure 5).
Among the 14 caves we studied, Gruta Olhos d' Água is remarkable by its high faunistic singularity, as shown by the high value of Taxonomic Distinctness. This value also indicates the presence of singular taxa as, for instance, troglobitic species (Cianciaruso et al. 2009, Gallão & Bichuette 2015. A relict species (Relictopiolus galadriel) recorded at Gruta Olhos d'Água corroborates environmental stability, enabling survival and evolution of species whilst its populations and sister groups outside caves were extinct by several factors as, for instance, climatic changes, competitive exclusion and stochastic factors.
The Lapa do Cipó cave presented the highest species richness, among which the troglobitic populations of I. uai, C. eleonorae (Monte et al. 2015), E. peruassuenssis (Bolfarini & Bichuette 2015) and X. odara (Campos-Filho et al. 2014). Besides the importance of its fauna, species richness is also high. Even more, these three troglobitic populations occur at Lapa do Cipó and Gruta Olhos d' Água caves, suggesting that both caves are part of a complex system. However, Lapa do Cipó showed a low TD value, probably because of its faunistic similarity with other caves.
In a study conducted at Chapada Diamantina, state of Bahia, a highest value of TD was recorded for the lowest richness cave, followed by the cave with the highest number of troglobitic species (Gallão & Bichuette 2015). In the region of Presidente Olegário, state of Minas Gerais, the highest TD value among seven caves studied is related to the presence of frugivorous guano patches and the presence of some animals that have a preference for this type of substrate such as Chilopoda of the genus Lamyctes (Zepon 2015).
Lower values of TD may indicate severe environmental degradation and pollution (Warwick & Clarke 1998). TD values at Caverna Cascudo, Caverna Boquete, Lapa dos Sonhos and Gruta Mina d' Água caves were significantly lower than expected at least for one of these,     Table 4 Caverna Boquete, we can state that this value may be related to anthropic impacts because of visitation. Non-significant values of TD were also found at caves from other localities. Zepon (2015), for instance, argued that the low value found at Gruta Juruva cave, Presidente Olegário, state of Minas Gerais, was due to low variety and quality of resources. In caves from Bahia, Gallão & Bichuette (2015) associated the low values of TD to extensive diamond mining in the past, which continues nowadays as a residual and clandestine activity. Our results show that contextualized studies of diversity are indispensable to conservation policies because, more than only characterize the fauna and spots of troglobitic occurrence, they also help the identification of complex subterranean systems and expand the potential for subsequent studies. Those discoveries are fundamental to conservation of the entire subterranean systems and its area of influence, a much more efficient approach to subterranean conservation than those focusing only in a given cave.
Maria Elina Bichuette -Substantial contribution in the concept and design of the study, contribution to data collection, contribution to data analysis and interpretation, contribution to manuscript preparation, contribution to critical revision, adding intellectual content.