Cryptic subterranean diversity in Collembola indicates that Hot Spot in ferruginous rock may have serious implications in Brazilian conservation policy.


 A super diverse hot spot of subterranean Collembola in ferruginous rock caves and Mesovoid Shallow Substratum is revealed by the analysis of cryptic diversity. The diversity is accessed by detailed description of chaetotaxy and slight variation in morphology of 11 new species of Trogolaphysa Mills, 1938 (Collembola, Paronellidae, Paronellinae) and the 49 previously recorded species of springtails from caves, using optical and electronic microscopy. When combined with recent subterranean surveys, our results show an important reservoir of cave diversity in the Mesovoid Shallow Substratum, contrasting with the conservation policy for subterranean fauna in metallogenic areas in Brazil which prioritizes the caves instead the cave species, which may be extremely detrimental to the fauna in the shallow subterranean habitats not accessible to humans.


Introduction
Some areas are subject of intense fauna diversification, the term "hot spot" is used to indicate relatively small areas with high and exclusive diversity, though there are different interpretations about what is the threshold which defines such an area. Hot spots may be defined by combining the richness, endemism, extension, and threats to the area in focus [1], however some approaches to subterranean fauna use an arbitrary cutoff of 20 restricted endemic species with no regards to environmental threats [2]. More recently two South American subterranean hot spots were defined based in the richness of restricted endemic fauna, and fully addressed the conservation aspects of the surroundings of the cave systems [3].
The species diversity of cave restricted fauna, mostly troglobites, is positively correlated to the extension of the cave and the presence of perennial pools, and sometimes negatively correlated to the presence of streams, which can cause disturbance in the habitats, and import a more diverse troglophile fauna [4]. Therefore, the more diverse troglobitic fauna is supposed to be found in larger caves, which are often formed in limestone rock, this is corroborated by the cave fauna hot spots found in limestone cave systems in Southeastern and Northeastern Brazil [3,5].
Nevertheless, there are subterranean spaces that extend through and across the weathered rock matrix, named Mesovoid Shallow Substratum (MSS) [6,7]. These underground spaces connect, and somehow extends the cave habitat far beyond the human reach and might be a climate refuge to epigeic fauna [8].
The Brazilian States of Minas Gerais (Southeast) and Para (North) represent the more important metallogenic areas in the country and concentrate the mining activities and commodities production. The iron ore lithology presents a profusion of small and shallow caves, subterranean spaces and crevices that functions as a real MSS [9], providing habitat for a variety of species, including troglobites [10,11]. Previous studies found higher average relative richness, and distinctiveness in ferruginous rock [9], than in other lithologies.
Unpublished data from caves and MSS in ferruginous rock, brings 87 undescribed species of Collembola with some degree of troglomorphism from Minas Gerais (73) and Para (14), a total of 37 species have been described so far (31 from Minas Gerais, six from Para), including 21 troglobites, among which 19 species in ferruginous cave and MSS (Table 1)

. Recent studies in
Brazil have surveyed hundreds of cave species, from sponges to vertebrates, more than 250 already described [3,11,12,13,14]. Great part of the subterranean biology research focuses on large caves in karstic lithology [15,16].
Here we present a group of 11 new species of Collembola of the genus Trogolaphysa Mills, 1938 with some degree of troglomorphism, from caves and MSS in ferruginous and limestone rock.
The genus Trogolaphysa has 69 described species worldwide, only eight have been recorded from Brazil: T. aelleni Yoshii, 1988 3 . *We consider troglobite all species with some degree of troglomorphism and known distribution restricted to subterranean habitats (for a discussion on troglobite definition see Sket [17].

Results
This study presents 11 new species of cave Trogolaphysa (Table 1), two new species from Para, eight from Minas Gerais from caves and MSS in metallogenic rock, and one new species from Sao Paulo, found in a limestone cave. Species were collected directly from organic debris in caves, the MSS was accessed through samplings in drilling holes. These results represent an increase of 24.8% in the previous 31 species of cave Collembola described for the State of Minas Gerais. Data from Para are still scarce, with only five previous records from iron rock caves and one from sandstone cave ( Table 1). The species from Sao Paulo is from a different lithology, a much larger cave with narrower connections to the MSS. It represents a new record to add to the 17 known cave springtails from limestone caves in Brazil (Table 1) With the results presented here the total number of cave Collembola recorded for Brazil rises to 60 species, with 18 species from limestone caves, one species from sandstone cave, one from granitic cave, and iron caves and MSS with 43 known subterranean species. The State of Minas Gerais present the highest richness for cave Collembola in Brazil, with two species from limestone and granitic caves respectively, and 37 records from iron caves and MSS. All these species are endemic to ferruginous rock shallow caves and MSS in Minas Gerais, this has important implications for the conservation areas policy in Brazil and other ferruginous rock subterranean environments in tropical areas in the world.

Ferruginous Mesovoid Shallow Substratum
The iron ore deposits in Brazil present a semi continuous covering layer of fragmented hematite and lesser components cemented by limonite, called Canga. It is formed by weathering and lixiviation, and produce a labyrinthic complex of subterranean spaces, crevices, and tiny underground connections, depicting a habitat that is analogous to the MSS [9].
In temperate zones the MSS plays a role as refuge for arthropod fauna, mainly at high altitudes where the cold weather can eliminate all the ectothermic fauna from the surface [8].
Similarly, seasonal migration movements are observed in the MSS for different taxa as response to hot dry summer [18,19]. When troglobitic fauna is concerned the MSS has a different role, cave restricted Collembola showed higher underground dispersal capacity than troglophiles [20], therefore, the MSS can connect neighboring caves systems and extend their distribution range.
In metallogenic rock, cave species richness is higher than in any other lithology [9]. The cave Collembola found in Brazil corroborates this assumption, from the total of 60 known cave species, one troglobite was recorded from sandstone caves and one from granitic cave, 18 species were recorded from limestone caves (all troglobites), and 43 species from iron caves and MSS (30 troglobites). Three troglobitic species were recorded from both limestone and iron caves, indicating potentially unrecognized cryptic species. This is more relevant when considered that ferruginous rock represents only 0.15% of the Brazilian territory, nearly 12,000 km 2 [21], and classic biospeleological research is focused on large (usually limestone) caves. The elevated richness of species restricted to small shallow caves, reinforces the role of the MSS as an extension of the cave environment, the diversity must be the result of local barriers related to the rocky outcrop and the surface phytophysiognomy.
The State of Minas Gerais is the most diverse with 39 species of cave Collembola, the complex mosaicist lithology and the ecotone Cerrado Forest-Atlantic Forest are the main barriers associated to the richness of species restricted to caves and MSS. In this State, the iron rock subterranean habitats host 26 troglobites, and provide habitat and refuge to 11 known troglophiles species.
For caves in non-ferruginous lithologies, the size and number of entrances influence the species richness by giving the surface fauna access to the subterranean environment, and as a sink for organic matter input [4]. Contrastingly the caves in iron rock are small and shallow, often with few meters of horizontal development, the connections to the MSS are visible and numerous. In this context, instead, the distribution of the troglobitic species suggests that the entrances of iron rock caves are the limits of the available subterranean habitat for troglobites inside-out, and of suitable habitats for troglophiles outside-in (Fig. 1). We can consider the entrances of these caves as windows of the MSS, the spatial limit of the subterranean environment which presents the minimum conditions to the survival of a troglobite, while partially inhibits the dispersion of troglophiles deeper in the MSS. Troglobites can disperse underground more efficiently than troglophiles, however troglophiles are more efficient than troglobites to disperse through the surface [20]. In ferruginous lithology the size of the cave and its entrance influences the species richness [4], mainly because large iron caves can greatly affect the capacity of collectors and biologists to access the troglobitic fauna in the MSS, as the number of accessible connections to the MSS increases exponentially with the length of the cave in iron rock [9].
Another contrast of ferruginous rock caves is that the biotrophic flow seems to be inverted ( Fig. 1), in limestone caves the energy and fauna come from outside through the cave entrance, and the fauna eventually speciate to become troglobitic, possibly restricted to the depths of a single cave. Despite the demonstrated existence of an epikarst, the particularities of the weathering process, and the water percolation [22], limestone caves tend to be large and grow deep through dissolution of the rock by water during the genesis of the cave. The deeper the cave is, the lesser the permeability of the rock, the epikarst usually reaches bout 15m deep [22].
In iron caves the fauna comes from the above ground through the MSS connections between surface and subterranean environments, the same happens with energy that comes with roots that reach the MSS abundantly [9]. The troglobites develop in the MSS and eventually reach the caves where it can be seen in its distribution limits, and the troglophiles go in the opposite direction, inhabiting the surface and going inside the caves to refuge from climate, but not going too far in the MSS (see Table 1, species marked with 1,3 ).

Figure 1.
Schematic profile of ferruginous rock cave and limestone rock cave. A) Ferruginous rock -small and shallow caves, abundant roots, reticulated MSS; fauna and energy come from the above ground (solid red arrow), troglobites inhabit the MSS and reach the limits of the cave, troglophiles inhabit the surrounding and the cave, eventually reaching shortly in the MSS (dotted red arrow). B) Limestone rock -large caves, usually not reached by roots, sparse or absent MSS; fauna and energy come through the cave entrance (solid red arrow), troglobites inhabit the deep aphotic zone reaching the aphotic intermediary zone, troglophiles inhabit the surroundings and the cave, eventually reaching the deep aphotic zone. Yellow to black bar represents the light reach.

Cryptic Diversity
Large caves with deep aphotic zones, stable abiotic conditions, water pools, often hosting bat colonies, are correlated to high number of restricted species [9], usually displaying classic troglomorphism as absence of eyes and body pigments, elongated appendages, increased body size [23]. In the ferruginous rock MSS the limited available spaces impose selective pressures that drive the troglomorphism to a different direction. Collembola species restricted to the MSS and caves in iron rock tend to be reduced in size, with normal to shortened (even though always functional) appendages, reduction in eyes number and body pigments.
There are discrete morphological differences in cryptic species, to access this information it is necessary to expand the morphological refinement, some species are grouped together as result of limited selection of diagnostic characters. Cryptic species recognized from a single widespread species complex through barcode sequencing, revealed related morphological differences corresponding to the species separation [24].
The species found in ferruginous caves and MSS are very similar in most of its macro morphology, the differentiation is masked by homoplasy, and species recognition often must rely on details of chaetotaxy ( Fig. 2-43) and slight variations of morphological structures, often overlooked, as observed for the genera Pseudosinella, Trogolaphysa, Arrhopalites, and Pararrhopalites. Such cryptic diversity can only be accessed by specialized scrutiny or molecular sequencing. Cryptic diversity in Collembola cannot be explained by accelerated rates of molecular evolution [25], therefore, the diversity of subterranean Collembola in ferruginous MSS and caves, possibly results of the combination of the effects of lithology arrangement, phytophysiognomy and climate fluctuation at local scale.
Finally, the recognition of cryptic species within presumed widespread allopatric species is crucial to efficiently develop management and conservation plans [20] and reduce the underestimation of cave Collembola diversity.

Subterranean Collembola Hot Spot
Brazil presents a total of 60 species of cave Collembola recorded so far, 39 are records from the State of Minas Gerais, including 28 troglobites (Table 1). Iron caves and MSS respond for 26 of those troglobitic species and provide habitat to additional 12 troglophiles. This is even more remarkable considering that, most of collection and research on cave fauna is directed to larger caves, usually in limestone.
Two important subterranean hot spots in Limestone caves in the States of Sao Paulo and Bahia (Southeastern and Northeastern regions), presented an overall species richness of 28 and 22 troglobites respectively. The two caves are under different impact pressures, the former is in a protected area with controlled access, and the latter is under intense touristic exploitation [3].
Myers et al. [1] combined richness, endemism, distribution spam and threats to the area to define places of priority for conservation, called hot spots. For subterranean diversity, eventually, an arbitrary number of troglobites is used to define hot spots, disregarding threats to the conservation [2]. The number of troglobites, with a full consideration of the threats or conservation conditions of the caves and surroundings, was recently used as criteria for defining hotspot [3]. Another hot spot for subterranean fauna is recorded from Rio Grande do Norte, Northeastern Brazil, they assumed the same criteria for defining the caves systems in the study as an "oasis of subterranean biodiversity" [5], where the diversity and threats were fully addressed.
The ferruginous rock outcrops in Brazil are under a ferocious economic pressure, the mining industry represents an important part of the production of commodities as iron ore and steel. The metallogenic deposits in Minas Gerais represent 0.15 % of the Brazilian territory, nearly 12,000 km 2 [21], the elevated diversity of cave Collembola found in recent studies, 26 troglobites and 12 troglophiles in iron caves and MSS (Table 1), the high degree of endemicity, considering these species are found nowhere else the beta diversity is directly impacted, and the continuous threat to the subterranean habitats formed in ferruginous rock, justify categorizing the ferruginous subterranean habitats as hot spot for cave Collembola in the State of Minas Gerais. It is important to remark that the diversity considered here is only for Collembola species, and that the studies mentioned above have a much higher phylogenetic diversity, but it may be an effect of the lack of sampling for other taxa, and the phylogenetic diversity of ferruginous subterranean fauna may prove to be much higher when all the fauna is accessed.

Conservation Policy Implications
The ferruginous caves and the MSS represent sites of intense cryptic diversification, Katz et al. [20] observed that, for Collembola the detection of short-range endemics, genetic isolation, and apparent cryptic diversity has major conservation implications. The combination of morphological and molecular techniques together can recover the cryptic diversity of Collembola in a complementary way [24].
The results we present here bring several considerations on conservation strategies and policies. The high diversity and endemism rate observed for cave Collembola, associated to threats to the subterranean environments as mining, deforestation, and urbanization flag these areas as maximum priority and interest for planning putative conservation areas [26]. These areas demand a multi-factor approach to successfully develop policies which optimize the diversity conservation, particularly subterranean diversity.
Brazilian legislation has protective measures for caves, but allows the complete suppression of a cave for mining or other exploratory purpose, under a process for licensing the proposed activities. Even though some criteria are imposed, it fails in considering some important aspects of the cave structure in different lithology [9]. Here we observed that the whole process needs a revision when comes to ferruginous rock, where the cave may not be the important spatial unit to preserve, instead, the high subterranean diversity areas must be surveyed, not only in caves but also in the MSS. It is possible that in some cases to protect a hill that harbors a thick layer of Canga with a troglobitic species rich MSS, would result more effective to preserve restricted subterranean fauna, than to protect a small and shallow cave with reduced troglobitic richness.
The state of Minas Gerais has 75 integral conservation units (defined by law), with maximum protection policy, however, these conservation units represent only 1.05% (~619,800 ha) of the state territory. There are other categories of conservation units, called of "sustainable use", with much less restrictive policies. These categories of conservation units are much less effective in order to preserve epigean species, due to the diverse usages and practices in those areas.
Nevertheless, the 19 sustainable use conservation units in the state of Minas Gerais (private conservation units excluded) correspond to 3.01% (~1,768,000ha) of the state territory (ief.mg.gov.br/unidades-de-conservacao -accessed Sep/31/2021). Sustainable use conservation units have some criteria that prevents highly destructive activities, allowing some extractive crops, subsistence agriculture and tourism. These activities may be compatible with subterranean conservation through the MSS, therefore the conservation units network can get some advantage trying to connect integral conservation units with sustainable use ones. It was proposed that the sampling for subterranean fauna in prospection drilling holes all over the area may bring important information about species richness and distribution, mainly if combined with cave and surface sampling [27]. This procedure, implemented in the process for licensing new high impact exploratory activities, can improve the conservation effectiveness of the conservation units and compensation areas, precisely define the role of the cave in the conservation plan, and shift the focus towards troglobitic species richness.

Conclusions
Our results depict the ferruginous subterranean environment as an important hot spot for cave Collembola in the state of Minas Gerais, corroborating the expected high species richness in ferruginous rock caves and MSS. We also demonstrate that access cryptic diversity as observed in the genera Arrhopalites, Pararrhopalites, Pseudosinella and Trogolahysa is mandatory for planning the conservation strategies for subterranean Collembola. The distribution of the species through the MSS can be favored by sustainable use conservation units, whether this fauna is surveyed along the licensing process. Finally, we conclude that the conservation planning for future conservation unit establishment must focus not only on caves but also in the MSS, accessing the fauna through sampling in prospection drilling holes. Protecting an area with high richness of endemic troglobites down in the MSS may be more effective than to protect a shallow cave when it comes to preserve troglobitic diversity.

Methods
Cryptic diversity. The richness was the measure of the subterranean diversity, we surveyed all data about previous records for Brazilian Collembola cave species, ecological status, lithology, and distribution from the literature, and included 11 newly found cryptic species from subterranean habitats in iron and limestone rock. The cryptic species were verified by comparison of chaetotaxy and "micro-morphology" through optic and scanning microscopy of disjunct populations of a widespread morphotype. The imagery was compared under hypotheses of chaetotaxic and morphologic homology, previously defined by different authors. Those populations with consistent discrete chaetotaxic and morphologic patterns were assumed to be independent species, therefore they were taxonomically diagnosed, named, and ordered in a dichotomic identification key with all Brazilian species of the genus, to test the validity of the species.
Microscopy. Specimens were preserved in ethanol 70% and mounted on slides following Jordana et al. [28], after clearing using Nesbitt's solution for study under phase contrast microscope, line drawings were made with help of a drawing tube. For Scanning Electronic Microscope (SEM) study, specimens were dehydrated by ethanol, dried in a critical point dryer, and covered in gold.