The first troglobitic Pseudochthonius Balzan, 1892 (Pseudoscorpiones, Chthoniidae) from the karst area of Serra do Ramalho, Brazil: a threatened species

Pseudochthonius ramalho sp. nov. is described to Gruna do Vandercir cave, in the Serra do Ramalho karst area, southwestern Bahia, Brazil. This area has an extensive limestone outcrop, with several caves, and the occurrence of potential minerals that are financially attractive for mining projects. The new species shows troglomorphic characteristics such as the depigmentation of the carapace and absence or reduction of eyes. It is a rare troglobitic species, and following the criteria of IUCN, we categorized the species as Critically Endangered – CR, IUCN criteria B1ab(iii)+2ab(iii). According to Brazilian legislation, locations, where critically endangered species live, can be protected by law, and we consider this cave/region to be of maximal relevance for protection.

, sheltering a high diversity (e.g., Trajano 2004, 2005;Bichuette and Rizzato 2012;Gallão and Bichuette 2018). It belongs to the Jacaré formation, Bambuí group, composed of dark, heterogeneous limestones, presenting intercalations with layers of claystone, deposited about 750 to 600 million years ago (Rubbioli et al. 2019). The region located in the middle of the São Francisco River basin is characterized by a tropical dry climate, with a dry winter and an average annual precipitation rate between 800 and 1000 mm (Gonçalves et al. 2018).

Material examined and treatment of specimens
Specimens were prepared by immersion in 85% lactic acid at room temperature for two weeks (Judson 1992). They were then examined by preparing temporary slide mounts with 10 mm coverslips supported by sections of nylon fish line (Harvey 2021). Specimens were examined with Nikon SMZ660 Stereomicroscope and Leica DMLS compound microscope and the male holotype was illustrated with the aid of a camera lucida. The female specimen was examined and illustrated through a Scanning Electron Microscope (SEM, FEI Quanta 250) in low-vacuum mode (ESEM) located at the "Instituto Nacional de Ciência e Tecnologia dos Hymenoptera Parasitoides da Região Sudeste Brasileira". After the study, the male specimen was cleaned in water and returned to 70% ethanol with its dissected parts in glass vials, and the female specimen was stored dry, due to the SEM picturing process.
Images (Figs 4,10) were taken with a Leica DFC 295 camera attached to a Leica M205C stereomicroscope with a Planapo 1.0× objective. Figures were produced from stacks of images on Leica Application Suite (LAS) software v3.7. The drawings were digitized and vectorized on Illustrator CC 2019. The maps were produced with the software QuantumGis Desktop 3.6.0 (QGis Open Source Geospatial Foundation). The coordinates were obtained from field trips to the study location with a global positioning system (GPSGarmin 60CSx).
The examined specimens are deposited in Laboratório de Estudos Subterrâneos, in Universidade Federal de São Carlos (LES, curator: Maria Elina Bichuette). For comparative purpose of some morphological characters like classical troglomorphisms in pseudoscorpions (eyes/ocular structures, proportionally longer body, and ratio pedipalpal chela/carapace), the new species was compared to two hypogean species, and one undetermined epigean species of Pseudochthonius sp.
Comparative material.
The following body structures were measured and compared proportionally for the characterization of possible troglomorphisms: carapace; chelal hand; chelal fixed finger (Christiansen 2012;Harvey and Wynne 2014;Feng et al. 2020;Harvey and Cullen 2020). Etymology. The species is named after the region of Serra do Ramalho due to its importance regarding the speleological heritage and the unique fauna and flora diversity. The name is to be treated as a noun in apposition.

Diagnosis.
Pseudochthonius ramalho sp. nov. can be identified by the following combination of characters: eyes absent (♂) or with eyes-spots (♀); the middle and distal fixed chelal finger teeth positioned two by two with 29-30 acuminate teeth and 3-4 slightly basally rounded ones, the presence of two rounded micro-denticles, along with pedipalpal fixed finger teeth in males; trichobothria ist closer to esb than to the est (ratio ist-est/ist-esb = 4.71); serrula exterior with 13 (♂) or 14 (♀) lamellae, rallum with seven blades, and coxae I and II with 3 to 5 coxal spines.  Description (adult ♂ and ♀). Body: Coloration of specimens in 70% ethanol yellowish brown and translucent pedipalps, tergites III-V with a dark median mark, and a darker abdominal region. Live specimens present a light pinkish color on their carapace and appendages, and a light brown abdomen. Female is slightly smaller than male.
Chelicera (Figs 6A, 7B, C): five setae on left hand, with one seta on the basal position of the fixed finger and one nearly the basal seta on the movable finger; six setae on the right hand; without the lateral microsetae; fixed finger with 10-11 (♂ and ♀) teeth proximally reduced in size; movable finger with 9 (♂) or 8(♀) teeth proximally reduced in size, three distal teeth distinctly larger than others and with subapical isolated tooth (di). Spinneret moderately prominent and apically rounded in female, vestigial in male. Seta gl 0.15 mm from base of movable finger. Serrula exterior with 13 (♂),14 (♀) lamellae. Rallum with seven blades pectinated. Dorsal face of cheliceral palm with four lyrifissures, three lyrifissures situated near seta dt and one situated posteriorly.
Pedipalp (Figs 6B-D, 8A-F): 1.4 (♂),1.2 (♀) × longer than carapace and 2.2 (♂), 2.6 (♀) × longer than patella; movable finger 1.6 (♂), 1.9 (♀) × longer than hand; fixed finger 1.65 (♂),1.72 (♀) × longer than hand. Fixed chelal finger long and strongly sigmoid in its distal half. Male fixed finger with 33 acute teeth, distinctly separated from each other, but paired and in each pair, one tooth is slightly directed to inside and the other to the outside, and micro-denticles in two interdental spaces, on teeth 15 and 29, respectively. Female fixed finger with 31 teeth arranged as in as in male. Mov- able finger with 30-33(♂ and ♀) flattened and separated teeth. Trichobothria: ib and isb situated close to each other sub-medially in the dorsal region of the chelal hand; eb closer to esb than to ist, forming a straight oblique row at the base of the fixed chelal finger; ist closer to esb than to the est (ratio ist-est/ist-esb = 4.71); et slightly near the tip of the fixed finger, near to the chelal teeth; dx, located near to the end of the fixed finger; sb closer to b than to st in the movable chelal finger (ratio sb-st/sb-b = 3.37); t closer to st and situated at the same level as est.

Distribution of the genus Pseudochthonius in Brazil
Species of Pseudochthonius occur in five Brazilian states ( Fig. 9): in the state of São Paulo (southeastern Brazil) with representatives of P. strinatii and P. ricardoi in cave habitats (Alto Ribeira karst area) and P. brasiliensis (in the region of Barueri); in state of Minas Gerais (southeastern Brazil), with the troglobitic species P. biseriatus endemic to the cave Olhos d'Água; in the state of Bahia (northeastern Brazil), with the new species described herein P. ramalho sp. nov., troglobitic and endemic of to Gruna do Vandercir cave, and also P. gracilimanus in cave habitat; in the state of Pará (northern Brazil) with representatives P. orthodactylus and P. tuxeni; and in the state of Amazonas (northern Brazil) the species P. homodentatus has been found in the Ducke Reserve and P. heterodentatus Hoff, 1946 was registered in the Urucu river basin (Aguiar and Bührnheim 1994). However, recently, this genus was recorded in other karst areas and biogeographical provinces, increasing its distribution to 37 more caves (Schimonsky and Bichuette 2019b).

Troglomorphic traits
Troglomorphic traits are characteristics that propose a relationship between hypogean species and the subterranean environment, associated with behavior, physiology, and mainly, morphology. Although these characteristics are useful to differentiate hypogean from epigean species, they do not explain the direct connection between the subterranean habitats and the species that inhabit it (Juberthie and Decu 1994).
Most families of pseudoscorpions have at least one troglomorphic feature. Chthoniidae can be considered one of the most important families regarding occurrence in subterranean habitats (Harvey et al. 2000;Reddell 2012). The most common troglomorphic characteristics are eye reduction and cutaneous melanin depigmentation, classified as regressive evolution (Christiansen 2012), in addition to progressive morphological changes, such as appendages elongation, which is fundamental for spatial orientation, defense, and predation in a habitat with the absence of light (Chamberlin and Malcolm 1960;Christiansen 2012). Examples of progressive morphological changes could be the comparison in the proportional length of the different body parts of different species, which highlights the appendages elongation, e.g., the tibia and the tarsus and of leg I. The hypogean species Pseudochthonius ramalho sp. nov. (5.2× longer than deep; 9.6-11× longer than deep), P. biseriatus (6.0× longer than deep; 10.3-11× longer than deep), P. strinatii (4.8× longer than deep; 9.5-10.7× longer than deep), P. ricardoi (5.3× longer than deep; 10.1× longer than deep). In the epigean species, P. heterodentatus (3.6× longer than deep; 10.0× longer than deep) and P. tuxeni (3.6× longer than deep; 7.3× longer than deep). Nevertheless, the epigean fauna of pseudoscorpions in South America is still little known (Mahnert and Addis 2002), which makes it difficult to compare hypogean and epigean individuals for the establishment of new troglomorphic characteristics. The female of the new species P. ramalho sp. nov. has ocular spots (Fig. 5) and the male has no ocular features. These characteristics should indicate a troglomorphic traits, that is, characters adapted to life in the subterranean environment (Fong 2012). Thus, these features can be compared with other Brazilian cave species (non-troglobitic), which have two small eyes (P. gracilimanus) or indistinct eye spots (P. ricardoi). When compared with some epigean species (P. thibaudi Castri, 1983 andP. arabicus Mahnert, 2014) it is noted that the absence or reduction of ocular traces is a troglomorphic trait (Fig. 10).
In the pedipalpal chela of the three hypogean species, there is a slight decrease in the length and width of the hand and a significant increase in the length of the fixed finger. These pseudoscorpions have, respectively, the following length and width: hand (in mm) -0.29/0.14 in P. ramalho sp. nov. (Fig. 11A1), 0.27/0.14 in P. strinatii (Fig.  11B1) and 0.26/0.13 in P. biseriatus (Fig. 11C1), and fixed finger (in mm) -0.59/0.04, 0.63/0.03 and 0.54/0.03, respectively. These values contrast with the ones observed in the epigean species Pseudochthonius sp.: hand (in mm) -0.31/0.19 and fixed finger (in mm) -0.49/0.04. Our observations corroborate that, for Chthoniidae, we cannot infer troglomorphism concerning to a single character (such as body pigmentation), but rather to a combination of traits (eyes/ocular structures, thinning of the cuticle, proportionally longer body, pigmentation, and ratio pedipalpal femur/carapace).

Conservation remarks
The Serra do Ramalho region is formed by several masses of carbonate rocks, thus enabling the occurrence of many karst features, including caves. Cave extensions range from hundreds of meters to more than 5 km, some exceeding 15 km (Rubbioli et al 2019). Another record of great importance is the great potential regarding subterranean fauna for both invertebrates and vertebrates (e.g., Baptista and Giupponi 2002;Pérez and Kury 2002;Bichuette and Trajano 2004;Bichuette and Trajano 2005;Trajano et al. 2009;Bichuette and Rizzato 2012). The issues related to the preservation of the subterranean environments in this karst region are directly influenced by the corresponding epigean environment. As the subterranean organisms use allochthonous organic matter, they rapidly suffer from the effects of any changes that occur to the epigean environment, e.g., deforestation and surface water pollution. Thus, studies demonstrate that the vast diversity of subterranean fauna is extremely important as an indicator of the health of the overall area (Bichuette et al. 2013;Gallão and Bichuette 2018). However, the Serra do Ramalho region is not yet inserted in any conservation units (e.g., State Park) and it is exposed to risks such as deforestation (e.g., wood for charcoal production), agriculture and mining projects due to the presence of rare metals (e.g., niobium) (Silva Junior and Campos 2016; Gallão and Bichuette 2018). The exploration and extraction of these metals can destroy entire caves and systems, leading to the extinction of isolated populations in these habitats (Culver 1986). This, coupled with the lack of laws that effectively protect caves in Brazil, leaves all this diversity of habitats under a high level of threat.
Pseudochthonius ramalho sp. nov. occurs exclusively in Gruna do Vandercir cave, being considered an endemic species to its type locality. By IUCN (International Union of Conservation of Nature) criteria, we classify this species as Critically Endangered (CR) according to criteria B1ab (iii) + B2ab (iii). This means that the species has a restricted geographical distribution, with an estimated occurrence of less than 100 km² (B1) and 10 km² (B2), and the severely fragmented population (a) lives in a few locations with the continued decline (b) in area, extension, and quality of habitat (iii). Therefore, effective protection measures must be taken so that there is no degradation of this environment, which is important in several aspects, and in this case, as the limited habitat of unique species that are very sensitive to disturbances.