On the enigmatic troglobitic scorpion Troglorhopalurus translucidus : distribution , description of adult females , life history and comments on Rhopalurus lacrau ( Scorpiones : Buthidae )

We describe for the first time the adult female of the troglobitic scorpion Troglorhopalurus translucidus Lourenço Baptista & Giupponi, 2004, and broaden its known distribution to other sandstone caves at Chapada Diamantina’s region. The life history of T. translucidus is reconstructed through morphometric measurements, with a multivariate analysis applied on a set of 16 specimens in all stages (including the holotype) and biological notes. The ontogeny of T. translucidus has seven stages and the litter size is 43 (n = 1). We discuss the isolation of T. translucidus and Rhopalurus lacrau Lourenço & Pinto-da-Rocha, 1997 in caves of Chapada Diamantina, and discuss their troglomorphisms (autapomorphies) among other species related with Rhopalurus. Both species must be considered fragile due their restricted endemism at Chapada Diamantina.

Anophthalmy, absence or reduction of the median and/ or lateral eyes, depigmentation, and reduction in sclerotization, are considered classical morphological troglomorphisms (Poulson 1963, barr 1968, barr & Holsinger 1985).The absence of a pectinal fulcra, reduction in the number of pectinal teeth, enlargement of the telson, absence of carapacial sutures and metasomal carinae, in addition to the shape and setation of the tarsi can be considered troglomorphisms in scorpions (Prendini 2001, volsCHenk & Prendini 2008).
The monotypic genus Troglorhopalurus Lourenço, Baptista & Giupponi, 2004, was created to describe a new troglobitic scorpion based on a single male that lacks pigmentation, from Lapão Cave, central Bahia, Brazil, a sandstone cave inside the Chapada Diamantina National Park, at altitude near 900 m.According to lourenço et al. (2004), this new genus features some phylogenetic affinities with Rhopalurus Thorell, 1876, mainly the presence and disposition of granulation on the fingers of the pedipalp.The most notorious divergence between these two genera is the absence of an stridulatory apparatus in Troglorhopalurus (lourenço et al. 2004).
In this paper we describe the adult female of T. translucidus, broaden its known distribution, give notes on the life history and biology of the species, and discuss troglomorphims and troglobitism with respect to the family Buthidae.dominated by karst landforms due to the dissolution of carbonatic rocks and, to a lesser extent, at its central portion, siliclastic rocks.The caves in the region, consequently, are formed by limestone and sandstone.Lapa do Bode Cave, the type-locality of R. lacrau, and Bob Cave (a new locality for the referred species) are limestone caves on the eastern border of Chapada Diamantina, and are located below 350 m (Fig. 1).These caves are inserted in Caatinga formation, and the climate there is semi-arid (BShw), according to köPPen (1948).The sandstone caves of Chapada Diamantina are located at higher altitudes than the limestone caves (above 500 m), and encompass the type-locality of T. translucidus (Lapão cave) and its new distribution record.The vegetation around those sandstone caves is formed by Campos de Altitude and Atlantic Forest remnants, with tropical semi-arid climate (Aw -tropical wet and dry), according to köPPen (1948).The limestone caves are warmer (temperature ca.28°C and relative humidity ca.95%) than the sandstone caves (temperature ca.20°C and relative humidity ca.95%), which may be related to their higher altitude.As mentioned above, the limestone caves are formed by rock dissolution, mainly carbonatic rocks, whereas sandstone caves are mainly formed by mechanical erosion of rock (Ford & Williams 2007); in the case of Chapada Diamantina, mainly by rivers.
Scorpions were hand collected, with the aid of UV light, and fixed in 70% ethanol.Some individuals were brought alive to the laboratory and were reared in terraria for further observations.Specimens of T. translucidus in different stages of development were collected from sandstone caves at Igatu, municipality of Andaraí, state of Bahia; R. lacrau were collected from Lapa do Bode Cave, Itaetê, Bahia; A total of 17 specimens of T. translucidus, five female adults, two female subadults and ten juveniles (of all stages) were collected from five sandstone caves at Chapada Diamantina, Andaraí, from 2010 to 2014.
The acronyms of the collections where specimens are deposited are as follows: MNRJ -Museu Nacional, Rio de Janeiro, Rio de Janeiro, Brazil; UFSCar -Universidade Federal de São Carlos, São Carlos, Brazil; LES -Laboratório de Estudos Subterrâneos.
The  Morphology and description -images of T. translucidus were taken with a Leica DFC 295 video camera attached to a Leica M205.Figures were produced from stacks of images using LAS (Leica Application Suite) v3.7.The ultraviolet images and photographs of live specimens were taken with 18.1 Megapixels resolution.The uncoated pectines of T. translucidus and R. lacrau were illustrated and analyzed through a Scanning Electron Microscope (SEM, FEI Quanta 250) in low-vacuum mode.
A multivariate analysis was performed to describe the ontogeny of T. translucidus, using Principal Components Analysis (PCA), since all data are quantitative (morphometric).Variables were tested by Shapiro-Wilk (p < 0.05) normality test and then log-transformed.We only used normally distributed variables and the PCA graphics were generated showing the dispersion of the operational taxonomic units (OTUs) on the sampling space.A matrix with 17 characters for 16 specimens, including the holotype, was constructed (Appendix 1).The characters used for the multivariate analyses were selected on the basis of structures that are relevant to scorpions and sum measurements were discarded.Shapiro-Wilk test and PCA were performed with the program PAST version 3.09 (Hammer et al. 2001).
Description (based on females and juveniles).Morphology: Scorpions 63-66 mm long: all measurements of the holotype and four female adults specimens of T. translucidus are recorded in Table 1.Prosoma (Figs. 2, 3): carapace slightly flattened dorso-ventrally; anterior margin with moderate median concavity with row of granulations; carapace with moderately marked granulation between anterior margin, lateral eyes and median eyes, lateral and posterior portions of carapace; portion between anterior median carina and between median eyes and central lateral carina with fine granulation; anterior median carina weak to moderately marked, lateral ocular, central lateral and posterior median carinae moderately to strongly granulated; central lateral carina starting almost at middle of lateral ocular carina; anterior median furrow moderately marked and central median and pos-terior median furrows strongly marked; median ocular tubercle slightly anterior to center of carapace, median eyes reduced and separated by little more than one ocular diameter; three pairs of lateral eyes.Chelicerae (Fig. 4): chelicera dentition characteristic of the family Buthidae (vaCHon 1963); dorsal granulation in one row; fixed finger with basal and medial teeth forming strong bicuspid; movable finger with two weak basal teeth, distal internal tooth slightly larger than distal external; ventral surface of fixed and movable fingers with long and dense setae.Mesosoma: tergites with weak to moderately marked granulation, with some larger granules at lateral-posterior region; presence of median carina on all tergites, weak to moderately marked; tergite I with median carina restricted to posterior area; tergites II-IV with median carina occupying distal half; tergites V-VI with median carina occupying more than distal half and almost complete on tergite VI; tergite VII pentacarinate with median carina occupying anterior region and moderately marked, submedian and lateral carinae strongly marked and submedian carinae complete and lateral carinae almost complete.Sternum subpentagonal; genital operculum divided longitudinally, each plate more or less suboval.Pectines (Fig. 5): pectinal tooth count 17-17 (n = 17 specimens, including the holotype); peg sensillae of pectinal tooth elongated and sharp (Figs. 16,17); fulcra present; basal middle lamellae of pectines not dilated; basal piece of pectines with two depressions.Sternites weakly granular; sternite III with two oval areas with weakly granulation without stridulatory apparatus (Fig. 5); in the sternites IV-VI granulation on lateral areas; sternite VII with complete weakly granulation; sternites IV-VI with two little depressions on central anterior region; setation moderate to strong; spiracles moderately elongate, width about twice lenght; sternites IV-V with very weak carinae; sternite VI with four moderately marked carinae occupying 3/4 of distal region; sternite VII with four strongly marked carinae, lateral carinae occupying central region and submedian carinae almost complete.Pedipalps: elongated; femur (Figs. 6, 7) with five carinae -dorsointernal, dorsoexternal, externomedian and ventrointernal continuous and crenulate and internomedian carina discontinuous with a row of spiniform granules; internal surface of femur with long setae, intercarinal surfaces finely and uniformly granular.Patella (Figs. 8, 9) with seven carinae -dorsointernal, dorsomedian, dorsoexternal, externomedian, ventroexternal and ventrointernal continuous and crenulate and internomedian discontinuous, comprising six moderately well spaced spiniform granules, becoming smaller in size distally; proximal tubercle moderately strong; internal surface of patella with moderately number of long setae; intercarinal surfaces very weakly granular, almost smooth.Chela (Figs. 10-13): with four complete or almost complete carinae (dorsal secondary, dorsal marginal, ventrointernal and ventroexternal), two vestigial carinae (digital and ventromedian) and three absent (dorsointernal, internomedian and subdigital); dorsal secondary and dorso marginal with thin granulation; ventroexternal complete with thin granulation; ventrointernal with thin granulation at proximal two-thirds and distal one-third without granulation; digital with few granules (about nine) at proximal region of chela manus, absent in the remaining chela manus; ventromedian with very few and thin granules (about five) at proximal region of chela manus, absent from the remaining chela manus; intercarinal surfaces almost smooth with very weak and thin granulation.Fixed and movable fingers (Figs. 10,11,13) very long and slender, weakly curved; internal and external surfaces of fingers with conspicuous setae; fixed and movable fingers with slightly oblique rows of granules, presence of internal and external accessory granules as well as presence of internal and external supernumerary granules; three granules at distal area of movable fingers, do not count as a oblique row of granules; fixed and movable fingers with moderately enlarged terminal denticle.Trichobothriotaxy: ortobothriotaxy A-α (vaCHon 1974(vaCHon , 1975)); trichobothrial counts: femur 11 (5 dorsal, 4 internal  -25), with chela manus and vesicle light reddish-brown, pedipalps reddish-brown and legs yellowish-brown.Juveniles and subadults depigmented.Prosoma (Fig. 2): carapace brown with dark brown spots surrounding median eyes region; reddish-brown on the anterior region of carapace and on the posterior edge; granulation of the anterior region reddish-brown and granulation of median and posterior region of carapace brownish; eyes with black pigment.Chelicerae (Fig. 4): reddish-brown with dark reddish-brown variegated spots over the entire surface; fingers reddish-brown; teeth dark reddish-brown.Mesosoma: dark brown zones on the anterior edge of tergites I to VI and light reddish-brown on the posterior edge of the same segments; tergite VII reddish-brown; granulation of tergite VII brownish.Coxosternal region reddish-brown.Sternites reddish-brown and sternites III-VI with light reddish-brown strip on the posterior edge.Pedipalps : femur and patella homogeneous reddish-brown, chela hand light reddish-brown, chela fingers reddish-brown at the base and light reddish-brown at the apex of chela fingers, granulation of pedipalps reddish to brownish.Metasoma (Fig. 14): segments I and II reddish-brown and segments III-V brown; granulation of all segments brown.Telson (Fig. 15): vesicle light reddish without spots; aculeus with light reddish at the base, reddish-brown medially and dark reddish distally.Legs: yellowish-brown with the tarsus of the legs whitish, ungues reddish.

Life history of Troglorhopalurus translucidus
A total of 17 specimens of T. translucidus were collected from five sandstone caves at Chapada Diamantina, broadening the distribution of the species to 40 km to the south (Fig. 1).lourenço et al. ( 2004) stated that this species was possibly an endemic element of caves from Lençóis, state of Bahia.Our new findings have revealed a wider distribution, with T. translucidus being apparently endemic to the central-north sandstone caves of Chapada Diamantina (Andaraí/Igatu in southern limit and Lençóis in northern limit).The sandstone of Chapada Diamantina is fractured, a result of having been formed by collapse/tectonism, erosion by streams and chemical dissolution (r.F.Pereira, pers.comm.).The connectivity between these spaces probably allows the dispersion of T. translucidus among different caves.
The ontogeny of females of T. translucidus has seven stages: the litter (not presented in PCA), stages 2 to 5 (juveniles), stage 6 (subadult) and stage 7 (mature) .Troglorhopalurus translucidus molts six times during its life, a common pattern for the family Buthidae (from four to seven) (Polis & sissom 1990).The stages of T. translucidus females can be easily recognized by quite distinct morphometric measurement and by the point dispersion in the PCA (Fig. 26).There is a great deal of homoplasy in toglomorphic organisms, for instance reduction of the eyes and melanic pigmentation, and behavioral traits (trajano & boCkmann 1999).Troglomorphisms are not necessarily adaptive, unless pleiotropic effects have been demonstrated (jeFFery 2010).Troglorhopalurus translucidus presents several morphological troglomorphisms, the most conspicuous being elongation of the appendages, mainly the pedipalps.Other morphological troglomorphic traits are the very long and slender fixed and movable fingers, attenuation of the carinae of the chela manus, reduction of the median eyes, attenuation of carapace granulation, reduction of pedal spur and shape of peg sensilla.The adult females of T. translucidus The most interesting fact concerning the ontogeny of females of T. translucidus is that the first axis is responsible for more than 98% of the variance in the PCA and the variables that most contribute in this first axis are length of metasoma I, II, III and IV, respectively (Table 2).Adding the second axis, almost 99.5% of the variance is explained and the variables that most contribute to this second axis are length of mesosoma, width of vesicle, posterior width of prosoma and length of metasoma I (Table 2).
Five adult females were examined, but only one male is known.Since it is known that sex ratio in Buthidae scorpions does conform to the 50:50 proportion and females are often oversampled (Polis 1990).
Comparing with the original description (lourenço et al. 2004) and with other examined juveniles, part of the morphological characters used to diagnose the male also apply to the females: body flattened dorsally, appendices long and slender, carapace flattened dorsally, median eyes reduced, pedipalps long 16 17 18 present pigmentation but the juveniles do not.Depigmentation is therefore not a troglomorphic trait of T. translucidus.It is important to document when juveniles are depigmented and adults are pigmented in studies that try to detect troglomorphisms, to avoid equivocal classifications, discussed for troglobitic centipedes by CHagas-jr & biCHuette (2015).

Rhopalurus lacrau
Since the discovery of the first cave animals, there have been many attempts to classify them into ecological-evolutionary categories (see trajano 2012 for review purposes).trajano (2012), proposed an alternative to the most accepted classification, as follows: trogloxenes -source population primarily in epigean environments; hypogean environment is used as part of the species' habitat, but the entire life cycle cannot be completed in it (hypogean); troglophiles -source populations in both hypogean and epigean habitats and generally maintaining the gene flow between them; troglobites -source population exclusively in hypogean environments, even when having sink populations in epigean habitats.Thus, cavernicolous species cannot be regarded as synonymous with troglobitic species, since the term cavernicolous encompasses these three.Although troglobitic species can exhibit some troglomorphisms, this is not obligatory and exclusive.
Despite the absence of conspicuous troglomorphic traits in R. lacrau, this species seems to be no longer capable to colonize (or recolonize) the epigean habitat.Searching for this species revealed no population or even solitary individuals outside the Lapa do Bode cave and a dead and dry specimen that probably belongs to R. lacrau was found inside Bob cave, about 20 km away on a straight line from Lapa do Bode cave (the hard parts of this specimen remain unaltered but the soft parts like pectin do not), which suggests that R. lacrau is vulnerable to desiccation (this hypothesis is under study).Caves are humid habitats and troglobitic arthropods in general have reduced cuticle (barr 1968).The current epigean habitat of the Itaetê region is composed of Caatinga formation with semi-arid climate (BShw), and relative humidity around 50% (pers.obs.).Alayotityus delacruzi is endemic to only one wet limestone cave in a semidesert coastal area in Cuba, and represents a troglobitic non-troglomorphic scorpion subjected to lower selection pressure, with regards to food, which is readily available (teruel 2001, teruel et al. 2006).The same applies to R. lacrau, since Lapa do Bode and Bob are wet caves inserted into Caatinga formation near epigean rivers of the Paraguaçu River Basin (Una and Paraguaçu rivers, Fig. 1).Lapa do Bode cave is frequently flooded by its epigean river (Nascimento pers.comm.).Whether this happens at Bob cave, we do not know.
An interesting question to answer is "how were the two species isolated in the caves?According to the reconstruction of H. Yamaguti, unpublished data, T. translucidus and R. lacrau share a common ancestor.As already mentioned, these two species share at least four morphological traits, including the shape of the peg sensilla, which could represent a shared troglomorphic feature .
The changes in the geomorphology and climate observed in the region in the last 30 Mya (Oligocene), including uplifting (tectonism phenomena) the sandstone over the limestone, 15 Mya (Miocene) evolution of the karstic system of Chapada Diamantina and, in the last 2 Mya (Pleistocene to nowadays), lowering of the base level, landslides, carving of Rio Una valley and capture of epigean rivers by subterranean drainages (R.F.Pereira, unpublished data), probably led to the isolation of the two species by vicariance.Greater selection pressure, including the scarcity of food in sandstone caves (gallão & biCHuette 2015) and an older isolation, which allowed for a greater accumulation of neutral mutations, could explain the presence of conspicuous troglomorphims in T. translucidus compared with R. lacrau.It is noteworthy that none of the two species can be as old as the Oligocene/Miocene periods, and consequently, the geomorphological changes in these periods are not relevant.Although the phylogenetic relationship between T. translucidus and Rhopalurus species remains unclear, T. translucidus and R. lacrau share, besides some morphological traits, the obligatory cave-dwelling life habitat.Thus, the number of troglobitic Buthidae is four species, Troglorhopalurus translucidus, Rhopalurus Alayotityus delacruzi and Troglotityobuthus gracilis (since we agree with lourenço 2000 about this species).The number of troglobitic species in Buthidae is low, only four species among more than 1,000 species (rein 2015).

Conservation remarks
The troglobitic fauna of Chapada Diamantina is remarkable.There are at least 23 troglobitic species in the sandstone caves, and more than 10 troglobitic species in the limestone caves of the same region, including beetles, centipedes, collembolans, scorpions, spiders, gastropods, fish etc (gallão & biCHuette 2015, M.E.Bichuette unpubl.data).In this region some relict taxa are isolated, for instance T. translucidus, as well as the only troglobitic tarantula from Brazil, Tmesiphantes hypogeus Bertani, Bichuette & Pedroso, 2013 and the recently described centipede Scolopocryptops troglocaudatus Chagas-Junior & Bichuette, 2015 in the sandstone caves of Chapada Diamantina.The same happens to the limestone caves such as Lapa do Bode Cave (the same with R. lacrau), where the highly specialized carabid Coarazuphium cessaima Gnaspini, Vanin & Godoy, 1998 and the ancient troglobitic catfish Rhamdiopsis krugi Bockmann & Castro, 2010 occur, plus some undescribed species.
Most sandstone caves of Chapada Diamantina were heavily impacted by diamond mining in the past, an activity that goes back to the early 1990's and, in a clandestine and residual way, continues to take place (biCHuette et al. 2008, M.E.Bichuette, pers.obs.).However, the sandstone caves where T. translucidus occurs are inside the Chapada Diamantina National Park and are, to some extent, protected.On the other hand, the limestone caves (Lapa do Bode and Bob Caves, locality of R. lacrau) are totally unprotected and can only count on the local population to protect them, with no support from environmental agencies.Currently, the problems concerning the limestone caves are deforestation of the surroundings areas for pastures, causing sedimentation of the Lapa do Bode Cave, lowering the water table due to agricultural irrigation, uncontrolled tourist visitation and pressure for limestone mining.Both species are listed as Endangered (EN), the second worst threat category, according to the last revision of the International Union for Conservation of Nature (IUCN).Studies such as this, on the distribution and life history of species, give support to the need for the effective protection of endangered species.

Figure 1 .
Figure 1.Distribution of Troglorhopalurus translucidus and Rhopalurus lacrau with type-locality and new records for both species.Question mark (?) indicates a new possible record in cave for R. lacrau, see text for explanation.

Appendix 1 .
Data matrix of the Principal Component Analyses (PCA) for T. translucidus.Measurements are in mm.

Table 1 .
Morfometric data of holotype and four adult females of Troglorhopalurus translucidus.The total length measure includes the telson.

Table 2 .
Results from the PCA for Troglorhopalurus translucidus collected in caves from Igatu, Bahia state.Eigenvalues on the left and eigenvectors on the right-hand columns, respectively.Bold type indicates characters that contributed for the most variation in PCA analyses.