Specimens were collected during inventories of cave fauna, which involved thoroughly investigating caves for invertebrates under blocks, in accumulations of organic matter, and in fissures in the soil or cave walls (Wynne et al. 2019). Additionally, a few occasional collections of specimens in epigean environment were performed. Specimens were collected with brushes and stored in containers with 100% alcohol.

Most of the material was studied as slide-mounted specimens. For this purpose, specimens were dissected, cleared in lactic acid and mounted on slides using Hoyer’s medium (Walter and Krantz 2009). Stomach contents were extracted from some specimens (n = 10) using micro-needles through a cut in abdomen. Fecal pellets were removed and mounted on microscope slides using hoyer. Some specimens (n = 12) did not require the removal of the stomach contents since clearing of the specimens was sufficient for analysis of the food items present in the fecal pellets.

For the terminology for the palp tarsal sensilla we followed Grandjean (1936) as modified by Vázquez and Klompen (2002), for the sternitogenital Sternitogenital region we followed Klompen et al. (2015), and for the leg sensory structures Grandjean (1936), van der Hammen (1966), and Araújo et al. (2018b). Morphological characters were processed in the data matrix development mode of vSysLab (Johnson 2010) and were exported as proto-species descriptions.

Drawings were prepared using a Leica MDLS phase contrast microscope (Leica Microsystens, Wetzlar, Germany), connected to a drawing tube. Measurements were taken from adults using an ocular micrometer and are presented in micrometers (μm), average length is presented first, followed by length range in parentheses. Photos were taken with a 3.2 mega-pixel digital camera attached directly to a microscope.

Collection sites of the specimens examined were georeferenced using coordinates in degrees, minutes and seconds with the World Geodesic System (Datum WGS84).

All specimens are deposited in the following collections:

MZLQ Coleção de Referência Acarologica, Universidade de São Paulo, Escola Superior de Agricultura ‘Luiz de Queiroz’, Departamento de Entomologia e Acarologia, Piracicaba, São Paulo, Brazil;

ISLA Coleção de Invertebrados Subterrâneos de Lavras, Universidade Federal de Lavras, Departamento de Biologia, Setor de Zoologia, Lavras, Minas Gerais, Brazil;

UFMG AC Acarological Collection at Centro de Coleções Taxonômicas, Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Belo Horizonte, Minas Gerais, Brazil.

Abbreviations: F = female, M = male.

Observations on seasonality were obtained from data produced by inventories of cave fauna in the municipalities of São Gonçalo do Rio Abaixo and Barão de Cocais, state of Minas Gerais, which used the method of active search as described above. The study was carried out in 109 cavities, with rainy season collections being made in January 2015 and dry season collections in October 2016.

Generalized linear models (GLMs), with contrast analysis were used to determine if there was a significant difference in the size of the Neocarus populations in the two seasonal campaigns. The models were built using abundance in each of the caves where Neocarus was observed as the response variable and each campaign as the explanatory variable. A negative binomial distribution with a log link functions was used since data presented significant overdispersion for Poisson error distribution (3.860). The GLM regression analysis was performed with R software (R Development Core Team 2019).

The ability to regenerate appendages can be a great advantage for species of Opilioacarida since these organisms easily lose their appendages during different stages of development or even in adulthood (Coineau and Legendre 1975; Klompen 2000; Bernardi et al. 2013a). As described in previous works (Coineau and Legendre 1975; Klompen 2000; Bernardi et al. 2013a), regeneration has been observed in young specimens of N. simmonsi, namely deutonymphs and tritonymphs. Here, we observed for the first time this replacement of appendages in adults. The specimen, a female, was collected while changing its integument, which made it possible to observe the old integument and the new integument formed below, with leg IV in regeneration (Fig. 10). Integumentary molting and continuous growth in Opilioacarida, even in adulthood, can be a physiological advantage for species of this group because they have a fragile integument, a possible autotomy behaviour and these mites can easily lose appendages.

Figure 10. 

Neocarus simmonsi sp. nov., view of regeneration process on leg (arrowed) in adult female (A, B); details of the molting process on chelicera.

It is not possible to say how many molts an adult can experience even after reaching sexual maturity, but the size of a male and female collected during field study suggests that growth may continue, and thus there can be more than one molt in adulthood. This is based on the observation that collected adult specimens exhibited the molting process, with two specimens (1 male and 1 female) having an old integument (exuvia), which was covering the new one. The new integument presented some characters already 9 to 18% larger than in any of the other adults collected. Based on the measurements of body setae, Bernardi et al. (2013a) suggested that there is a continuous increase in adult body size after molts. Vazquez and Klompen (2002) observed a greater number of setae in super-adults, but this morphological change was not observed in the specimens studied here.

Another interesting observation made on an adult male of N. simmonsi was the presence of morphological abnormalities of the palp, with the reduction and reorganization of setae as well as changes in the shape of the structure itself (Fig. 11). Small abnormalities in body setae (Araújo et al. 2018a) or the duplication of pores (Bernardi and Borges-Filho 2018) has been observed in other species. Here, for the first time, abnormalities are seen in complete structures, such as the palp. The potential behavioral consequences for individuals remain unclear.

Figure 11. 

Neocarus simmonsi sp. nov., detail view of the morphological abnormality on palp of male.

The gut contents of specimens of N. simmonsi includes unidentified plant fragments, arthropod remains, pollen, and fungal hyphae (Fig. 12). The observation of plant fragments and pollen in the stomach contents of specimens collected in the deepest areas of caves (more than 40 m from the entrance to aphotic zones in some caves) suggests that these organisms feed on decomposing matter from the epigean environment that is transported by water action. Three specimens of N. simmonsi had also specimens of mites, possibly of the family Tarsonemidae and Oribatida. This feed plasticity can assist in the colonization of a great diversity of habitats, including caves, where resources generally tend to be scarce.

Figure 12. 

Example images of the gut content of individuals Neocarus simmonsi sp. nov., A, B exuvia of Opilioacarida, C mite (Astigmatina), D pollen, E probably a bothridial seta (oribatida), F plants, fungi and arthropods parts, G mite exuvia, probably an Endeostigmata.

In addition to these materials, the stomach contents of three specimens evaluated under microscopy possessed remains of integuments (exuvia) of Opilioacarida (Fig. 12). When reviewing the stomach contents of other specimens of other species present in the collect of Universidade Federal de Lavras (ISLA), we also observed materials of this nature in Caribeacarus brasiliensis (4 individuals), N. caipora (3 individuals), N. proteus (4 individuals) and other undescribed species of Opilioacarida (8 individuals).

During the observation of N. proteus kept laboratory condition in 2012, for about 4 months, also allowed documenting specimens feeding on their own exuvia after molting (2 observations) and even exuvia of other dead individuals (2 observations). Such laboratory data, associated with the observations of stomach contents of specimens of N. simmonsi in the present study and the absence of records of cannibalism among Opilioacarida bred and maintained in the laboratory (Vázquez and Palacios-Vargas 1989; Klompen 2000; personal observations), allows us to infer that these species feed on their own exuvia. This behavior is not unique among arachnids, but is apparently rare. Some observations made by Dondale (1965) suggest that spiders feed on exuvia because they contain a set of proteins that can be an important source of nutrients for individuals after going through the stress of molting, and the period without energy intake. This is an interesting behavior, because it allows Opilioacarida to reabsorb energy present in the tissue or liquid, discarded in the process of ecdysis. This behavior can be an important strategy in an environment with scarce resources, such as some caves.

Neocarus simmonsi does not have morphological characteristics arising from isolation in subterranean environments and was found both in caves and the epigean environment. The only species whose morphology has been modified to the point of being considered troglobitic are Siamacarus dalgeri and S. withi, described by Leclerc (1989) from caves in Thailand. As adaptations to the subterranean realm, these species lack eyes, possess elongated appendages, have a pale coloration, and even modified sensory setae. Similar traits have not been found in cave species in Brazil thus far.

The distribution of N. simmonsi extends across the entire Serra do Tamanduá and Dois Irmãos (municipalities of São Gonçalo do Rio Abaixo and Barão de Cocais) in a strip of at least 12 km. However, the effective distribution of the species is expected to be much greater, considering that it is a common species in the sampled area and that it is not restricted to subterranean environments (caves) (Figs 13, 14).

Figure 13. 

Distribution of Neocarus simmonsi sp. nov..

The study area is located northeast of the Quadrilátero Ferrífero (Iron Quadrangle), in an area of Atlantic Forest near the transition zone with the Cerrado biome (Brazilian savanna). The vegetation cover consists of forest formations and “campos rupestres” (rupestrian fields) at higher altitudes and shallow soils rich in metals (e.g. iron). Since it is a geomorphologically diversified area and located close to the transition between two biomes, there is a great diversity of flora and fauna. The Sternitogenital region, therefore, is considered a priority area for conservation in the state of Minas Gerais with “extreme biological importance” due to the high floristic and faunal richness and the presence of several endemic and endangered species (Drummond et al. 2005). There are a few hundred registered caves in the study area including the largest caves in the Quadrilátero Ferrífero.

Figure 14. 

General aspects of Simmons’ cave (MDR_0020), (A, B) and epigean environmental (C), habitat of Neocarus simmonsi sp. nov.

The type locality, Simmons' cave, was mapped in 1960 and is one of the few examples of caves formed by dissolution in the Sternitogenital region. The cave is located at approximately 1140 meters above sea level and has only one entrance, aphotic zones, different compartments (halls), perennial lakes, and 146 meters of horizontal projection. With regard to organic resources, litter deposits were observed in the entrance area, along with guano of carnivorous and hematophagous bats. Lichens and fungi were observed on the walls and floor near the entrance to the cavity.

Neocarus simmonsi is an abundant species in caves in the Sternitogenital region, having been found in 73 of the 109 studied caves. The abundance of specimens was significantly higher in the wet season (estimated β ± S.E. = 1.29328 ± 0.22096; z = 5.853; p-value = 4.83e^–9), ranging from few to dozens of individuals as the case of BRU_0012 cave (n = 48 during the wet season and n = 0 during the dry season), BRU_0023 cave (n = 21 during the wet season and n = 1 during the dry season), and BRU_0002 cave (n = 15 in the wet season and n = 1 during the dry season). This great oscillation may be explained by the fact that the number of individuals observed increases during the rainy season, or else it may be due to the migratory behavior of the species (Fig. 15). Even in caves, where the environment is generally stabler than at the surface, seasonality can still influence the general climate (Bento et al. 2016; Mammola and Isaia 2018), and decrease the availability of water and food (Simon et al. 2007; Silva et al. 2011; Souza-Silva et al. 2012). Nevertheless, further investigations are needed to verify the generality of this pattern.

Figure 15. 

Average sampled individuals of Neocarus simmonsi sp. nov. per cave according to seasonality. The abundance values are significantly different between the two seasons according to the Generalized linear model analysis (z = 5.853; p-value = 4.83e^–9) In the boxplots, the turquoise areas refer to the interquartile range around the observed median (central black line) and vertical bars represent the maximum-minimum range (excluding outliers).

In some caves individuals of N. simmonsi were found aggregated in groups of up to eight and ranging from protonymph stages to adult males and females. The species’ behavior is similar to that observed for N. caipora and Caribeacarus brasiliensis (Bernardi et al. 2014). As suggested by Vázquez et al. (2018), this gregarious behavior can increase the survival rate, especially for young individuals. Neocarus simmonsi was significantly more abundandant during the wet season (Fig. 15), when there is greater rainfall in the Sternitogenital region (above 1000 mm). Besides climate, another factor that may determine variations in the abundance of this species is the availability of organic resources. Organic matter is imported into caves by biological agents (e.g. bats), wind or rain (Simon et al. 2007; Silva et al. 2011; Souza-Silva et al. 2012), with this transport being generally greater during the wet season.

The number of described species of Opilioacarida from collections in karst areas and caves in Brazil is increasing (Bernardi et al. 2013b, c, 2014; Araújo et al. 2018a; Bernardi and Borges-Filho 2018). This is due to the intensification of sampling efforts in this habitat as the result of environmental licensing processes. Environmental licensing requires speleological studies to be carried out in karst areas or other sites that potentially bear caves in Brazil among them the inventory of cave fauna (BRASIL, 6.640, IN MMA 2017). This legal requirement has lead to the discovery and description of several new species in the country, in addition to the acquisition of information on the range and biology of various species.

Brazil stands out for its diversity of described species of Opiliacarida (11 spp.) and its great potential for new records in view of its territorial extension and the large concentration of caves in different lithologies and biomes (Lewinsohn and Prado 2005; Oliveira et al. 2016; CECAV 2019). Thus, an increase in the number of new species is expected with intensification of sampling effort in different ecosystems and greater investment in taxonomic research and training of new specialists.

Mites and other soil invertebrates generally exhibit seasonal fluctuations, with their richness and abundance being determined by environmental factors such as precipitation and temperature (Badejo 1990). Although caves are confined systems and have greater environmental stability than the surrounding epigeal environment (Culver 1982; Freitas and Littlejohn 1987), the cave microclimate can also influence the structure and composition of fauna, with fluctuations in populations and communities throughout the year. Such variation is often a response to variation in the external climate (Ferreira et al. 2015; Bento et al. 2016), as indirectly observed for N. simmonsi.

Opilioacarida is a group of species with interesting feeding habits. The material ingested by them is composed of large fragments of solid material of vegetable, animal and/or microbiological origin (van der Hammen 1966; Walter and Proctor 1998; Klompen 2000; Araújo et al. 2018a; Bernardi and Borges-Filho 2018). Although small invertebrates (especially mites) have been observed in the stomach contents of N. simmonsi, it is still not possible to say whether species in this group are predators or scavengers. For example, our observations of the stomach contents of N. simmonsi would suggest that these organisms are one of the few known “scavengers” of Parasitiformes, but this remains an anecdotal observation.

Finally, the order Opilioacarida always occupies a prominent position when referring to the study of development among mites. The development of these taxa comprises an embryonic phase (prelarva), subsequent larva, protonymphs, deutonymphs, tritonymphs and adults, but its growth can continue beyond the adult phase. The regeneration of appendages in adult individuals, however, is a peculiarity known only in this group among Parasitiformes and is rarely observed among a few Acariformes and arachnids in general (Michener 1946; Imamura 1952; Furumizo and George 1976; Bernardi et al. 2013a). Growth in adulthood can result in individuals with larger bodies and morphological structures and a greater number of setae in some areas of the body (Vázquez and Klompen 2002; Bernardi et al. 2013a). In the present study, we observed that growth and regenerative ability occur in all life stages of Opilioacaridae, which may eventually increase their survival rate.