Pseudonannolene canastra sp. nov. (Diplopoda, Spirostreptida) – a new troglobitic millipede from the southwestern state of Minas Gerais, Brazil

Pseudonannolene is a neotropical genus of millipedes distributed in Brazil, Argentina, Paraguay, Uruguay and Bolivia. Generally, species of Pseudonannolene are considered troglophilic, i.e., they can establish a source population in both subterranean and superficial habitats. Among the 60 species known, 49 are found in Brazil; out of these, 25 occur in caves but only three are considered troglobitic (source population exclusively subterranean). This study aims to describe the fourth troglobitic species of Pseudonannolene from Brazil, and the first one from the region of Serra da Canastra, in the southeastern part of Brazil.


Introduction
Pseudonannolene Silvestri, 1895 (Spirostreptida, Pseudonannolenidae) is a neotropical genus of millipedes distributed in Brazil, Argentina, Paraguay, Uruguay and Bolivia (Schubart 1944;Mauriès 1987;Enghoff et al. 2015). The diagnostic feature of this genus is the presence of a longitudinal division of the promentum of the gnathochilarium (Enghoff et al. 2015). Generally, species are considered troglophilic (Trajano et al. 2000;Enghoff et al. 2015), i.e., they can establish a source population in both hypogean (subterranean) and epigean (superficial) environments with individuals that can move between these habitats, thus promoting the flow of selected genes in both environments (Trajano 2012;Trajano and Carvalho 2017).
Among the 60 species known, 49 were reported in Brazil and 25 among these occur in caves, while three are considered troglobitic (Iniesta and Ferreira 2014) i.e., the source population is exclusively subterranean (Trajano and Carvalho 2017). Most of the described species are in the states of Minas Gerais and São Paulo (Gallo and Bichuette 2019). In Brazil, the known troglobitic species include P. spelaea Iniesta & Ferreira, 2013, from Pará state, P. ambuatinga Ferreira, 2014, andP. lundi Iniesta &Ferreira, 2015; these last recorded in the Minas Gerais state. For these species, the troglomorphisms, i.e., modifications in morphological characteristics associated with subterranean isolation (Christiansen 1962), include reduction in body pigmentation, variation in number of ommatidia, body size, and antennae elongation (Iniesta and Ferreira 2015).
Despite little knowledge about this group, two cave species, P. ambuatinga and P. spelaea, are included in the List of Brazilian Threatened Fauna under Critically Endangered (CR) and Endangered (EN) categories, respectively (Chagas Jr et al. 2018a, b). This classification is based on the IUCN (International Union for Conservation of Nature) rules and adapted to the Brazilian criteria.
This study aims to describe the fourth troglobitic species of Pseudonannolene from Brazil and the first one from the region of Serra da Canastra in southeast Brazil and discuss its troglomorphisms and conservation status.

Study area
The Gruta do Tesouro cave ( Figure 1) is located in the municipality of São Roque de Minas, in the region of Serra da Canastra, Minas Gerais state, 350 km away from the capital Belo Horizonte. The climate of the region, according to Köppen's classification, is Cwa with dry winters and warm summers (Alvares et al. 2013), with rainy season occurring from October to April and dry season from May to September. The vegetation is composed of Cerrado (savanna-like) with riparian and mesophilic forests (Batista et al. 2018).
The Gruta do Tesouro cave is composed of limestone rock belonging to the Bambuí geomorphological unit and has 1,320 m of passageways ( Figure 2A). The cave is outside the Serra da Canastra National Park (PNSC) and its surroundings are affected by the pastures adjoining the cave entrance ( Figure 2B); uncontrolled tourism is another factor impacting the cave-dwellers. The specimens occur on the riverbank substrate in an aphotic zone, i.e., in complete darkness ( Figure 2C) with bat guano piles close by.

Collections
The specimens were manually captured by active search and placed in a non-toxic plastic container and taken to the Laboratório de Estudos Subterrâneos at Universidade Federal de São Carlos (LES/UFSCar). The specimens were observed daily for seven days and photographed alive at laboratory. The specimens were then fixed in 70 % ethanol and photographed. The images were captured on a Leica DFC295 camera coupled to a Leica M205C stereomicroscope with Planapo 1.0× lens and produced by mounting multiple images using the LAS software (Leica Application Suite) V3.7. The holotype was analyzed using a Scanning Electron Microscope (FEI Quanta 250, FEG-SEM) in a low-vacuum mode. All individuals that did not present pairs of legs in all segments of the body, that is the growth zone, were considered juveniles (Makarov 2015). The gonopods and the first leg-pair of the male were drawn with the program Adobe Illustrator CC 2014 and the map prepared in the QGIS program version 3.4. The studied material was deposited in the zoological collection at the LES / UFSCar. We examined the species deposited at the Museu de Zoologia da Universidade de São Paulo (MZUSP) (listed in Table 1); besides, the original descriptions of all species of Pseudonannolene (eg. Schubart 1944Schubart , 1945Schubart , 1947Schubart , 1949Schubart , 1958Schubart , 1960Silvestri 1895Silvestri , 1897Silvestri ,1902Bröle-

Taxonomy
Diagnosis. Pseudonannolene canastra sp. nov. has wide coxa of gonopods resembling those of P. microzoporus Mauriès, 1987, while its solenomerite shape is similar to that of P. maritima Schubart, 1948. However, P. canastra sp. nov. differs from these species in characteristics such as the round shape of the gonopod coxa, the telopodite with a wide base, reduced dentiform processes, different number of spines in the inner part of the coxa (three in the left coxa and four in the right coxa in caudal view), and a divergent pre-femoral process with reduced and flaky pre-femur prolongations. Externally, these species are quite distinct. Pseudonannolene microzoporus, considered troglophilic, has a uniform light brown pigmentation (Mauriès 1987) while P. maritima, recorded only in epigean environment, is brownish or brownish-black, and the head and the first two segments are always lighter, with nearly yellowish color (Schubart 1948).
Holotype description (male). Body length 36.26 mm, ring diameter 1.98 mm, with 59 rings (counting the collum and the telson) ( Table 2) without anterior or posterior tapering of the body; the prozonite is shorter (0.04 mm) than the metazonite (0.33 mm); metazonite has thin striae in the ventrolateral portion of all the rings; the gnathochilarium is typical of the genus Pseudonannolene with longitudinally divided promentum (Enghoff et al. 2015); head has a posterior suture from the collum to the line of the eyes. Antenna: Slender (2.3 mm length), covered by fine white setae almost transparent, with some setae larger than others; antennomeres longer than width; the third antennomere is the largest; the sixth antennomere is the widest with a round shape and four sensory cones in the end. The antenna length ratio to the head 1.7 (from the beginning of the suture from labrum) while diameter ratio 1.17.
Eyes: oval-shaped lateral eyes area 0.09 mm², lengths 0.44 mm and 0.43 mm (right and left, respectively); width 0.3 mm; twenty-two ommatidia (Richter et al. 2010) on the right side and 24 ommatidia on the left side (Table 2).
Gonopodium ( Figure 6A-D): The coxa is round, 0.45 mm long and 0.34 mm wide; the dentiform processes reduced in number and size ( Figure 3A) with three internal spines (or processes, Iniesta and Ferreira 2013) on the left side and four on the right side ( Figure 3B, one of the spines is not visible in the figure); the solenomerite end rounded and scaly ( Figure 3C) (0.24 mm long, 0.12 mm wide), corresponding to 0.54 and 0.35 of the coxa length and width, respectively; the telopodite smaller than the solenomerite in length (0.122 mm) but larger in width (0.139 mm) and corresponds to 0.51 and 1.17 of the solenomerite length and width respectively; it measures 0.15 mm in length and has a wide base (with thin setae) that narrows gradually ( Figure 3D).
First pair of legs ( Figure 6 E-F): coxa longer than wide (0.58 mm length, 0.28 mm width); setae are concentrated in the distal region of the coxa and few setae are found in the proximal region (Figure 6 D); the coxa base is slightly narrow; the prefemur wider (0.18 mm) than length (0.15 mm); strong setae concentrate on the inner side of the pre-femur; pre-femoral process divergent, as small, rounded and scaly extensions in the base of the pre-femur ( Figure 3E); femur (0.27 mm), post-femur    (0.15 mm), tibia (0.13 mm), and tarsus (0.22 mm) with thick, elongated setae; the tarsal claw is 0.15 mm long. Walking legs (mean values): total length 1.79 mm; coxa (0.21 mm), pre-femur (0.26 mm), femur (0.33 mm), post-femur (0.27 mm); tibia (0.23 mm); tarsus (0.34 mm) with setae on the inner side; all podomeres longer than wide; the tarsal claw 0.14 mm length.
Telson: The anal valve has 2 + 2 setae, no sulcus; the pre-anal sclerite does not extend beyond the anal valve; the subanal plate lacks projection (Table 2).
Observations. The juveniles, i.e. individuals that did not present pairs of legs in all segments of the body (Makarov 2015), show symmetry in the number of ommatidia; eyes are more triangular than oval. The other characteristics of the body are the same as those of the adults. For this species, the females are larger than the males.
Adult millipedes generally have dark pigmentation, while the juveniles can be lighter because of the ecdysis process (Makarov 2015). However, P. canastra sp. nov. maintains light pigmentation in adult stage. The reduction of integument pigmentation was observed in the troglobitic species P. ambuatinga, P. lundi, and P. spelaea. Darkening of the pigmentation of P. canastra sp. nov. was also observed when they were exposed to artificial lighting. This darkening has already been reported for P. spelaea (Iniesta and Ferreira 2013).
Some cave-dwelling species of the genus Plusioglyphiulus (Spirostreptida, Cambalopsidae) present ommatidia varying from four to 13; this was not considered as a troglomorphic trait . Some examples of troglobitic species belonging to the order Spirostreptida are Cambala speobia Chamberlin, 1953; C. reddelli inornatus Causey, 1964;and Mexicambala russelli Causey, 1964; they have been identified from caves in North America. These species show troglomorphic traits such as lack of eyes (C. speobia and M. russelli) or reduced number of ommatidia ca. 2-7 (C. reddelli inornatus). The species of the Pseudonannolenidae family have four to 45 ommatidia; they are rarely absent (Enghoff et al. 2015). Following this pattern, we do not consider that P. canastra sp. nov. has a reduced number of ommatidia. Besides, ommatidia are absent in the pseudonanolenid Typhlonannolene adaptus Chamberlin, 1923, an epigean species, which reinforces the idea that the reduction (until its complete absence) in the number of ommatidia is not simply related to the isolation in the subterranean environment. Therefore, it is difficult to infer if this character-state truly represents a troglomorphism.
There are some gaps in the description of the Pseudonannolene species. One of them is related to the antenna measurements. Forty-one percent of descriptions do not mention the exact measurement or only report that the antenna is long. For the remaining 59% of the described species, the ratio between the antenna length and the body diameter ranges from 0.59 for P. maritima to 1.55 for P. tricolor -the last species is considered troglophilic. Considering the troglobitic species -P. spelaea, P. ambuatinga and P. lundi, the ratio ranges from 0.86 to 1.04 (P. ambuatinga), 1.03 to 1.08 (P. lundi), and 0.93 to 1.06 (P. spelaea). The antenna length of 2.3 mm in P. canastra sp. nov. is 1.17 of its body diameters, the largest for the troglobitic species. However, this ratio is also large in the troglophilic species P. microzoporus (1.27), P. strinatii (1.23 to 1.5) and P. tricolor (1.52 to 1.55), for example, even larger than that observed in the troglobitic species.
Considering the body dimensions, the order Spirostreptida ranges from 0.6 to 20 mm in body diameter (Enghoff et al. 2015). The troglobitic species Cambala reddelli inornatus, M. russelli and C. speobia have a body diameter of 1.0, 1.3, and 1.9 mm, respectively. For the Pseudonannolenidae family, the body is considered slender compared to the other spirostreptidians, with diameters varying from 0.7 to 6 mm (Enghoff et al. 2015); most of the Pseudonannolene species have a diameter ranging from 2 to 5 mm. The adult P. canastra sp. nov. shows a body diameter of 1.97 mm and is thinner when compared with the other two troglobitic species P. ambuatinga (2.4 to 3.36 mm), and P. lundi (2.4 to 2.8 mm). However, it is slightly larger than the troglobitic P. spelaea (1.74 to 1.88 mm) in body diameter. Some troglophilic species like P. leucocephalus and P. taboa show their body diameter ranging from 1.4 to 2.2 mm and 5.5 to 6.1 mm respectively. So, we cannot infer whether the body diameter observed in P. canastra sp. nov. is a reduction and consequently a troglomorphism or not.

Endemism and conservation
The Serra da Canastra is known for its scenic beauty with large rocky walls, waterfalls, and its biological richness of fauna and flora (ICMBio 2018). However, the Serra da Canastra National Park (PNSC) is the only protected part of this region. The Gruta do Tesouro cave is outside the perimeter of the PNSC and located on private property, therefore, it is unprotected. The farm owners manage tourist activities, and no management plan is implemented.
Pseudonannolene canastra sp. nov. is the first troglobitic species described for Serra da Canastra. The present species is geologically isolated from the other species described for the genus Pseudonannolene due to their occurrence inside the caves. The Municipality of São Roque de Minas is in the geomorphological unit of Bambuí, specifically in the formation of Paraopebas (Karmann and Sanchéz 1979). The nearest described species are from the Pains region located about 85 km from São Roque de Minas, inserted in the Sete Lagoas formation (another formation of the Bambuí geomorphological unit) (CODEMIG 2003).
Pseudonannolene canastra sp. nov. is mostly found in a specific substrate (the bank of a river) with high humidity; any change in its habitat is dangerous. This species is endemic to the Serra da Canastra region and restricted to the Gruta do Tesouro cave, which is threatened by uncontrolled tourism and deforestation for pasture. Samplings in other caves in the region were made and this species were not found. This can be justified by the limited dispersal power of millipedes (Hopkin and Read 1992;Golovatch and Kime 2009) resulting in a high degree of speciation, and the evolution of many endemic species (Hopkin and Read 1992). The troglobitic species described were found in very moist microhabitats in the caves, associated with drainage or speleothems and, bat guano and vegetable debris as food resources (e.g. Iniesta and Ferreira 2015). Because of these conditions, we carried out a preliminary evaluation concerning the status of conservation of this species following the IUCN (International Union of Conservation of Nature) classification. Pseudonannolene canastra sp. nov. was classified as Critically Endangered (CR) with the criteria B1ab (iii): B being the restricted geographical area; B1 the occurrence extension of the taxon (<100 km² according IUCN, the present species has 51.9 km²); a represents one location and b(iii) represents the continuous decline of habitat quality. In addition to P. canastra sp. nov., two more subterranean species are known from the Serra da Canastra (different taxonomic group) that has been sampled by LES team (Laboratório de Estudos Subterrâneos) since 2009 (Gallão and Bichuette 2018). This shows the potential and biospeleological importance of the region, as well as the need for adequate protection and management of the cave, its surroundings, the watershed and landscape (Gallão and Bichuette 2018). and 310378/2017-6; regular project -457413/2014-0) and Coordeação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, process 88887.159166/2017(CAPES, process 88887.159166/ -00 / project number 440646/2015 for financial support to MEB; ICMBio (Instituto Chico Mendes de Conservação à Biodiversidade) for granting the license (28992-7); the Angelica Maria Penteado Martins Dias for the use of the Stereomicroscope and Scannig Electron Microscope (SEM). (INCT Hympar Sudeste -Process FAPESP 2008 / 57949-4 andCNPq 573802 / 2008-4); Luciana B. R. Fernandes for the photos obtained through the stereomicroscope and Scannig Electron Microscope (SEM).; Grupo Bambuí de Pesquisas Espeleológicas (GBPE) for permission to use the cave map; CS Fernandes for help in the field work; MZUSP (R Pinto-da-Rocha and M. Cardoso) for the loan of the type material; DM von Schimonsky for help in the field, preparation of the map and donating the photos; to DJ Tomaz for donating the photos; and CS Fontanetti, CS Fernandes, JE Gallão and T Zepon for their suggestions and help during this study. We would like to thank Editage (www.editage.com) for English language editing. We thank the reviewers William Shear and Ivan Tuf for the careful suggestions made to the manuscript.