Four new troglophilic species of Loxosceles Heinecken & Lowe, 1832: contributions to the knowledge of recluse spiders from Brazilian caves (Araneae, Sicariidae).

Four new species of recluse spiders from Brazilian caves are described with both males and females. Loxoscelesericsoni Bertani, von Schimonsky & Gallão, sp. n. and L.karstica Bertani, von Schimonsky & Gallão, sp. n. both occur in caves in the Peruaçu region, located in the northern area of the state of Minas Gerais; L.karstica sp. n. is additionally found in the Serra do Ramalho karst area, located in the southwestern region of the state of Bahia. These two species belong to the gaucho group. Loxoscelescarinhanha Bertani, von Schimonsky & Gallão, sp. n. and L.cardosoi Bertani, von Schimonsky & Gallão, sp. n. occur exclusively in caves of the Serra do Ramalho karst area and belong to the rufescens/amazonica species group. The discovery of two additional and highly distinct species in the rufescens/amazonica group (L.carinhanha sp. n. and L.cardosoi sp. n.) increases the debate on the origin, evolution, and geographical distribution of this widely distributed group of recluse spiders in the New and Old World. The presence of three species (L.ericsoni sp. n., L.carinhanha sp. n., and L.cardosoi sp. n.) with marked differences in morphological characters in a relatively small area indicates that the region seems to be an important center for Loxosceles diversity, which remains poorly studied.

species of Loxosceles in Brazil, and only four, L. adelaida, L. similis, L. willianilsoni, and L. troglobia (the only troglobitic species in Brazil), occur in caves. However, it is worth mentioning that the majority of the records of Loxosceles in Brazilian caves are still at a generic level (Trajano 1987, Pinto-da-Rocha 1995, Cordeiro et al. 2014, Gallão and Bichuette 2015. Worldwide, this genus has been recorded in caves in Iran, Thailand, South Africa, and Namibia (e.g., Chomphuphuang et al. 2016, Tahami et al. 2017, Lotz 2017. It is also noteworthy that, for some caves in Namibia and South Africa, there are records of at least seven species, two of them coexisting in one cave in South Africa (L. parramae Newlands, 1981 andL. speluncarum Simon, 1893) (Lotz 2017).
The aim of this paper is to describe four new Loxosceles species with a discussion about distribution and diversity of this genus in Brazilian caves.

Study sites
Studied regions are in the transition zone of the Cerrado and Caatinga morphoclimatic domains (Ab'Saber 1977), and, according to the Koppen-Geiger classification (Peel et al. 2007), the climate is tropical semi-arid, with a well-defined dry period between April and September and an average annual temperature of 24 °C and a maximum rainfall of 800-1000 mm (INMET 2017).

Peruaçu region, in the northern area of the state of Minas Gerais in southeastern Brazil
The Janelão, Bonita, and Boquete Caves (Figs 1-3) are located in Peruaçu Caves National Park (PCNP), in the state of Minas Gerais in southeastern Brazil, and are under legal protection. The region is covered by extensive limestone outcrops of the Bambuí geomorphological group (Piló and Kohler 1991) and is home to the richest  cave in Minas Gerais, Olhos d'Água Cave, with at least 12 obligatory cave species of troglobites (Trajano et al. 2016, Gallão andBichuette 2018).

Serra do Ramalho karst area, state of Bahia, northeastern Brazil
The Serra do Ramalho karst area (Figs 1,(4)(5)(6) comprises several limestone outcrops of the Bambuí geomorphological group, including several large cave systems, some reaching several kilometers in length (Auler et al. 2001). The region is one of high subterranean diversity, and there is no legal protection for this region (Trajano et al. 2016). The main threats are deforestation for agriculture and pastureland, in addition to potential mining projects for cement production and mineral products (Gallão and Bichuette 2018). Gertsch (1967) was used as the basis for species descriptions. Structures from the left side of the specimens were used, or, when the right side was used, the figures were mirrored to show them as coming from the left side to allow for easy comparison. A Leica LAS Montage and LAS 3D module mounted on a Leica M205C dissecting microscope were used for image capture and measurements of spider structures. Left legs and palps were measured from the dorsal aspect of the left side. All measurements are in millimeters. The copulatory organs of females were dissected and digested with a commercial protein remover for contact lenses (with pancreatin) for several minutes in order to observe the internal structure; when necessary, they were also cleared with clove oil.

Abbreviations:
ALE anterior lateral eye, PLE posterior lateral eye, PME posterior median eye.
Etymology. The specific name is in honor of Ericson Cernawsky Igual from Grupo Pierre Martin de Espeleologia (GPME) for his contribution to Brazilian speleology and his commitment to the conservation of caves.
Remarks. Loxosceles ericsoni sp. n. females have highly modified spermathecae (Figs 15-17). Although they have a transverse plate, it is very narrow and does not connect directly to the two receptacles (Figs 16,17). The receptacles themselves are two long, slender tubes positioned parallel to the transverse plate and converging to the center. Despite the modified female genitalia, it is possible to include this species in the gaucho group by the male palpal morphology, as they have a cymbium almost the same length as the palpal tibia (Figs 11,12). Diagnosis. Males of Loxosceles karstica sp. n. resemble those of L. ericsoni sp. n. by the palpal tibia length more than 1.4 and less than 2.0 times the cymbium length. They can be distinguished from those of L. ericsoni sp. n. by the shorter cymbium and stouter embolus (Figs 26,27). Females of L. karstica sp. n. resemble those of L. similis, L. chapadensis, and L. niedeguidonae by the spermathecae having the sclerotized transversal plate with two long and straight receptacles. They differ from the females of the species above by the short, sclerotized transverse plate with the receptacles positioned at an angle of 45° to the inner side (Figs 20,21). Additionally, they can be distinguished from females of L. chapadensis by the dorsal part of the bursa copulatrix medially sclerotized ( Fig. 21) and from L. niedeguidonae by the non-incrassated palpal tarsus (Fig. 19).
Remarks. The male specimen in better condition to be chosen as paratype lacks legs I and III. For this reason, another male (MZUSP 74434), not in condition to serve as type, had legs measured as follows: leg I: femur 8. The new species L. karstica sp. n. has genitalic characteristics shared with other species of the gaucho group distributed in the southern regions of Brazil and Paraguay, as L. gaucho, L. similis, L. adelaida, and L. variegata, which have palpal tibia that are short and incrassate in males (Figs 26,27) and the large spermathecae transverse plate in females (Figs 20,21). It also shares characteristics with the species of the gaucho group that are more distributed in the northern part of Brazil, as L. chapadensis and L. niedeguidonae, as they have a longer palpal tibia in males and a spermathecae transverse plate that is almost as short as in the new species but with differences in the bursa copulatrix sclerotization and the size and shape of receptacles.
Etymology. The specific name refers to karst, a word used to define terrain with distinctive hydrology and landforms that arise from a combination of high rock solubility and well-developed porosity. Loxosceles karstica sp. n. occurs in two important karst areas of Brazil (Peruaçu and Serra do Ramalho).   Gallão leg., 29.v.2012 (MZUSP 74439).

Loxosceles carinhanha
Diagnosis. Males of Loxosceles carinhanha sp. n. can be distinguished from those of all other Loxosceles species by the thick embolus (Figs 29-31), a strong curvature on basal metatarsus I, and a constriction on distal tibia I (Figs 34, 35). Females of L. carinhanha sp. n. resemble females of L. cardosoi sp. n. by having spermathecae as a large, weakly sclerotized pouch with two large receptacles on its distal portion. Females of L. carinhanha sp. n. can be distinguished from those of L. cardosoi sp. n. by the spermathecae lacking a sclerotized transverse plate and dorsal parts of bursa copulatrix having only a small sclerotized triangular area (Figs 39-41).
Description. times as long, tibia I 1.8 times as long and leg I 7.4 as long as carapace. Palpal femur 5.6 times longer than wide; tibia 2.0 times longer than wide; cymbium oval (Figs 32, 33). Bulb suboval and slightly larger than cymbium. Embolus thick and straight, with a curvature on apex, approximately 1.3 times longer than bulb length in retrolateral view, without carina (Figs 29-31). Femur I prolateral median area with a series of enlarged setae (Figs 34, 36). Metatarsus I strongly curved on its basal portion. Distal tibia I abruptly narrow, with a series of strong macrosetae before the constriction (Figs 34, 35). Cephalic region of carapace, fovea, and thoracic striae with long, greyish setae (Fig. 28). Carapace and chelicerae uniformly reddish (Fig. 28). Abdomen, legs, and palp light brown, covered by short, greyish setae. Coxae and sternum light brown; labium and endites slightly darker.
Etymology. The specific name refers to the type locality of the species, Carinhanha, a municipality in the southwestern section of the state of Bahia, Brazil. The region possesses several cave systems with high diversity and a fragile subterranean fauna.
Remarks. Loxosceles carinhanha sp. n. and L. cardosoi sp. n. males have a uniformly reddish carapace (Figs 28, 42) instead of the brown marked carapace characteristic of the groups gaucho and rufescens/amazonica, and femur I has macrosetae on its prolateral median area (Figs 36, 48), which is exclusive of the two species. They occur in closer areas and are probably sister species. The inclusion of the two species in one of the groups defined by Gertsch (1967) for South American Loxosceles is not simple question. They could fit in either gaucho or rufescens/amazonica groups. Males of gaucho group have the cymbium and tibia subequal in length (Gertsch 1967). However, two species described more recently has slightly longer and slender tibia (L. chapadensis and L. niedeguidonae). Even though the tibia is not incrassate in these species, the cymbium is larger than the bulb, projecting forward. Considering the variation of tibia length and width in this group, we consider the cymbium size a better character to diagnose males of gaucho group. Males of the rufescens/amazonica group have the cymbium considerably shorter than tibia. More important, however, is they are never much more larger than the bulb. Based in this criterion, both L. carinhanha sp. n. and L. cardosoi sp. n. can be included in the rufescens/amazonica group (Figs 32, 33, and 46, 47). Concerning females, those of the gaucho group are readily recognizable by "the seminal receptacles attached to immovable, sclerotized, transverse plate" (Gertsch 1967). We noted that in species of gaucho group the receptacles are always slender and strongly sclerotized, except the apex and can be another diagnostic character. Those of the rufescens/amazonica group have the "seminal receptacles with a cluster of small, globular lobes at apex" (Gertsch 1967). More recently, at least two species were known to have a single large lobe at apex, L. mahan Ribera, 2015 from Canary Islands andL. willianilsoni, from Brazil (Fukushima et al. 2017). We consider that the main characters shared by females of rufescens/amazonica group is the spermathecae triangular shape, two free receptacles (not attached to a transverse sclerotized plate) with large basal transverse openings with or without sclerotized edges and two dark sclerotized lateral bands with distinct levels of sclerotization depending on the species (see Ribera 2015 andFukushima et al. 2017 for spermathecae variation). Loxosceles cardosoi sp. n. females have a transverse sclerotized plate (compatible with those species of gaucho group) and the receptacles are short (contrary to rufescens/amazonica group) and broad (as in the rufescens/amazonica group). A single dark sclerotized band is present (another characteristic of rufescens/amazonica group). The bursa copulatrix is strongly sclerotized. The putative sister species, L. carinhanha sp. n. has spermathecae weakly sclerotized lacking a transverse sclerotized plate and the receptacles are free. The bursa copulatrix is weakly sclerotized, except for a central triangular area. In favor of the inclusion of L. cardosoi sp. n. and L. carinhanha sp. n. in rufescens/amazonica group are the short cymbium in males and the broad and no sclerotized receptacles in females. Additionaly, L. carinhanha sp. n. spermathecae have a single dark sclerotized band. There is no supporting character for the inclusion of males in the gaucho group. In females, L. cardosoi sp. n. has the characters transverse sclerotized plate and short receptacles, which are lacking in L. carinhanha sp. n. Therefore, it seems more parsimonious to include the two species in the rufescens/amazonica group, elevating to five the number of species of this group in South America. These two species are very distinctive of the other species of the group both in the New and the Old World.
It has been proposed the origin of Loxosceles rufescens group in the Old World with a posterior introduction of L. amazonica during portuguese colonization of Brazil beginning in 1500 (Duncan et al. 2010). One of the evidences for the introduction hypothesis was the lack of other related species in South America (Duncan et al. 2010). Recently, Fukushima et al. (2017) described two species related with L. amazonica and L. rufescens from Brazil and argued contrary to this possibility for the short time (500 years) for speciation taking place. The discovery of two additional and very distinctive species reinforces the proposal of Fukushima et al. (2017). As the Loxosceles diversity in South America is still largely unknown, it is necessary more efforts to collect and describe species from more remote areas of Brazil, mainly those in the northeastern and central western regions, as the areas under study here, which seems to be a hot spot for Loxosceles diversity. Diagnosis. Males of Loxosceles cardosoi sp. n. resemble those of L. carinhanha sp. n. by having a group of macrosetae on the median prolateral area of femur I (Fig.  48). They can be distinguished from the males of L. carinhanha sp. n. by the slender embolus with a gentle curvature on its median area ending in a strong curvature on its apex  and the straight metatarsus I. Females of L. cardosoi sp. n. resemble those of L. carinhanha sp. n. by having spermathecae as a large, weakly sclerotized pouch with two large receptacles on its distal portion. Females of L. cardosoi sp. n. can be distinguished from those of L. carinhanha by the spermathecae having a sclerotized transverse plate, one dark sclerotized band reaching the basal area of each receptacle and dorsal and ventral parts of bursa copulatrix strongly sclerotized (Figs 51-54).
Etymology. The specific name is in honor of Dr. João Luiz Costa Cardoso, a physician who worked for several years at the Hospital Vital Brazil, Instituto Butantan, São Paulo, Brazil, treating bites and stings of venomous animals and publishing several related articles.
Remarks. See remarks under L. carinhanha sp. n.

Discussion
According to Trajano and Carvalho (2017), troglophilic populations are easily found more in subterranean habitats than in epigean habitats, probably by the differences in species dynamics. Generally, troglophiles present higher densities in subterranean habitats and low densities on the surface and they can move between them (Trajano and Carvalho 2017). The presence of individuals at all ages in subterranean habitats is one of the strongest pieces of evidence for troglophilic populations, contemplating such distribution along the years including different annual cycles.
Autapomorphic states, known as troglomorphisms in troglobitic species, evolving because the subterranean habitats (by natural selection, neutral mutation or even pleyotropy) are relevant clues to state if a species are obligatory and exclusive cavedwelling, however, this method is only valid when used in a comparative method.
Loxosceles karstica sp. n., L. carinhanha sp. n. and L. cardosoi sp. n., only found inside caves, do not show any troglomorphisms, at least morphological, such as elongated appendices, reduction of visual organs, sclerotization degree or pigmentation, when compared with other Loxosceles species, as observed in L. troglobia, the only troglobitic representative of Loxosceles in Brazil (Souza and Ferreira 2018). It is noteworthy that epigean habitats for these species are very dry, mainly in the winter season in Brazil, which may lead not to find these species in the surface, however, individuals of this new Loxosceles species may be encountered in suitable habitats on epigean surface. Presence of troglomorphisms do not define troglobitic species, but in most cases are cause of the incompatibility of troglobitic species living in epigean habitats (Trajano and Carvalho 2017), as seen in L. troglobia.
The presence of three new species (L. ericsoni sp. n., L. carinhanha sp. n., and L. cardosoi sp. n.) with very distinct morphological characteristics in a relatively small area (Fig. 1) indicates that the regions of Peruaçu and Serra do Ramalho are important centers for Loxosceles distribution, which remains poorly studied.
The karst areas of Peruaçu and Serra do Ramalho have different conservation statuses. Peruaçu's caves are under legal protection as part of a National Park (Peruaçu Caves National Park-PCNP), and part of its cave fauna is included in the Brazilian RedList (at present, four species); by contrast, the Serra do Ramalho karst area has no legal protection, and the main strategy to protect its karst, caves, and cave fauna is to use the Brazilian RedList, since six of the 14 troglobites are included on this list (Gallão and Bichuette 2018). Both areas are included in the governmental program for priority areas for conservation (PAN), but until now, no action has been proposed. The main threats to both regions are deforestation in Itacarambi and Januária and deforestation together with mining projects in the Serra do Ramalho karst area (Gallão and Bichuette 2018). Although the new species of Loxosceles described herein are not obligatorily cave-dwelling, they are endemic to two regions with a high degree of endemism of troglobites and are part of a fragile community, representing a strong argument for protection of these regions' cave fauna.