New troglobitic species of Niambia from Botswana and Namibia (Crustacea, Isopoda, Oniscidea)

Three new species in the genus Niambia are described from southern African caves: N. botswanaensis sp. nov. from Diviner’s Cave (Botswana), N. ghaubensis sp. nov. from Ghaub Cave and N. namibiaensis sp. nov. from Märchen Cave (Namibia). All these species show troglomorphic traits such as absence of body pigmentation and eyes. These are the first troglobitic species recorded in the genus. Most of the other species of Niambia are epigean and occur in semi-arid environments in the Afrotropical Region.


Material and methods
The specimens were manually collected and fixed in 70% ethanol for preservation. Measurements and photographs were taken with a ZEISS Axio ZoomV16 stereomicroscope coupled with an Axiocam 506 Color camera at the Center of Studies on Subterranean Biology of the Federal University of Lavras (CEBS-UFLA, Lavras, Brazil). Body parts were mounted in micropreparations using Hoyer's medium. Drawings were made with the aid of a camera lucida on Leica DM750. The noduli laterales were measured and illustrated as proposed by Vandel (1962) and the illustrations were prepared using the software GIMP (v. 2.8) with methodology proposed by Montesanto (2015Montesanto ( , 2016. For analysis of the dorsal cuticular structures and some body parts, pictures were taken with the scanning electron microscope Hitachi TM4000. Holotype and paratypes of the new species were deposited in the collections of Subterranean Invertebrates of Lavras (ISLA) in the Federal University of Lavras, Brazil, and in the Museo di Storia Naturale dell'Università, Sezione di Zoologia "La Specola" (MZUF), Florence, Italy. Description. Maximum length: male, 3 mm; female, 3.5 mm. Colorless (Fig. 1A). Dorsal surface of pereon sparsely granulated with scattered triangular scale-setae (Fig.  1E, F); one line of noduli laterales per side on pereonites, close to posterior margins and at certain distance from lateral margins, b/c and d/c co-ordinates as in Fig. 2A; gland pores not visible. Cephalon ( Fig. 1B) with suprantennal line visible only at sides, no frontal line, eyes absent. Pleon slightly narrower than pereon, pleonites 3-5 with falciform epimera. Telson (Fig. 2B) triangular with straight sides, obtuse apex. Antennula ( Fig. 2C) with three articles, second article shortest, distal article with eight short aesthetascs. Antenna (Figs 1B-D, 2B) reaching distal margin of pereonite 3 when extended backward; fifth article of peduncle and flagellum subequal in length; flagellum with two articles, second article about 3 times as long as first. Mandibles (Fig. 2E, F) with molar penicil consisting of 5-6 setae arising from common stem; left mandible with 2 + 1 penicils, right mandible with 1+1 penicils. Maxillula (Fig. 2G) outer branch with 4 + 5 teeth (three of them slightly cleft) plus one small tooth between outer and inner teeth; inner branch with two short penicils. Maxilla ( Fig. 2H) with bilobate apex, inner lobe distinctly smaller than outer lobe. Maxilliped (Fig. 2I) basis rectangular; palp basal article with two stout setae; endite rectangular, medial portion with one long seta, distal margin with one tooth. Pereopod dactylus with inner claw stout, slightly longer than outer one; pereopod 1 ( Fig. 3A) carpus with transversal grooming brush. Pleopod exopods without visible respiratory structures. Uropod (Fig. 2B) protopod reaching distal margin of telson, slightly grooved on outer margin; endopod and exopod inserted at same level, endopod distinctly shorter than exopod.
Etymology. The new species is named after Botswana, where the specimens were collected.
Taxonomic remarks. This and the following new species are included in the genus Niambia since they show all the characters of the genus, except for lacking respiratory areas in the pleopod exopods. This condition may be due to a secondary reduction due to the humid environment of the caves where they occur. Most of the species of Niambia are epigean and inhabit semi-arid habitats in South West Africa (Barnard 1932). In lacking the frontal line on cephalon, N. botswanaensis sp. nov. resembles N. duffeyi Taiti, 1981 andN. longiantennata Taiti &Ferrara, 1991 from Ascension Island, as well as N. septentrionalis Taiti & Ferrara, 2004 from Socotra Island (Yemen). However, it is distinct in lacking pigmentation and eyes, and in the shape of the male pleopod 1 exopod without posterior point (compare fig. 11 in Ferrara and Taiti 1981;fig. 30 in Taiti and Ferrara 1991;and fig. 33D in Taiti and Ferrara 2004).
Some specimens of this species from the type locality were sent to Dr. M. Javidkar in Adelaide for molecular analysis and were included in a phylogenetic cladogram as Niambia sp. 1 (Javidkar et al. 2015).
Ecological remarks. The Koanaka and Gcwihaba hills are located in the North-West District (or Ngamiland) of Botswana. The lithology was described by Williams et al. (2012) as Precambrian dolomites from the Damara Sequence. According to Thies and Lewis (2015) the surrounding environment (known as the Kalahari Thirstland) is semi-arid with shrub savanna vegetation and forms part of the Savanna Biome. Annual precipitation typically ranges between 400 and 500 mm and temperatures from -8.5 °C to 42.2 °C.
The Koanaka and Gcwihaba hills collectively host four known caves with natural entrances, which include Gcwihaba Cave, a local tourist attraction formerly known as Drotsky's Caverns (Fig. 4A). Furthermore, using gravimetric surveys and exploration drilling, an additional five caves were discovered, opened and explored (Harvey and Du Preez 2014;Du Preez et al. 2015). One of these is Diviner's Cave, the type locality of Niambia botswanaensis sp. nov. Initial exploration of Diviner's Cave commenced in 2011 after a 700 mm diameter vertical shaft (borehole) was drilled ( Fig. 4B-E). This shaft is 41 m deep and enters the cave in a chamber called 'Entrance Alcove' (Fig. 4C, D). Upon initial exploration of the cave, high CO 2 levels were encountered, especially in areas lower than the general cave development level. However, since the cave was allowed to vent following the drilling of the borehole, CO 2 levels gradually decreased and facilitated further exploration. As can be expected, atmospheric conditions in Diviner's Cave and those with man-made entrances were quite distinct from the observed in local caves with natural entrances (Du Preez et al. 2015). All the caves as- Specimens of Niambia botswanaensis sp. nov. were collected by hand in Calcite Baboon Chamber, Diniver's Cave. The collection site is located (Fig. 4F, G) at the one end of this chamber where large Ficus cordata roots penetrate the cavity (Du Preez et al. 2015). Associated with this root system is a community of soil invertebrates that include pseudoscorpions (Botswanoncus ellisi Harvey & Du Preez, 2014), diplurans (Japygidae), centipedes (Cryptops sp.) and termites.
Taxonomic remarks. Niambia ghaubensis sp. nov. differs from N. botswanaensis sp. nov. by the telson with right-angled instead of obtuse apex, male pereopods 1-2 with a brush of setae on carpus and merus sternal margin instead of sparse setae, dactylus of pereopods with a thinner inner claw, and male pleopod 1 exopod cordiform instead of rounded.
Ecological remarks. The Ghaub cave is the third largest cave in Namibia, with approximate 2.5 km of passages. It consists of an intricate net of labyrinthine conduits with different levels. The lowest level connects to the phreatic level; thus, some ponds are observed inside the cave. The cave is inserted in the dolomites of the Otavi geological group, dating from the upper Precambrian (Goudie and Viles 2015). The cave has a single entrance (Fig. 8A), in which there is currently a gate installed to prevent unauthorized entry. The conduits' morphologies are somewhat variable since they transect an intricate stratigraphy along the almost 40 m vertical gap within the rock (Fig. 8B,   Figure 8. Niambia ghaubensis sp. nov. A Ghaub cave's entrance B cave intricate stratigraphy C cave conduits D specimen of Niambia ghaubensis sp. nov. E cave chamber with mud animals F external hills surrounding the cave. C). Although the lower levels of the cave are connected to the phreatic level, most of the cave remains dry. Yet, moist substrates occur in some areas by drip water from speleothems. Organic resources observed are mainly the guano produced by insectivorous bats, where invertebrates are usually concentrated. Specimens of Niambia ghaubensis sp. nov. were only found in the deep sections of the cave, associated with moist substrates (Fig. 8D). Curiously, such areas were devoid of other invertebrates, and also of guano. Hence, it seems that this species is highly specialized, avoiding areas richer in organic matter and, thus, other invertebrate species (including predators). Individuals were observed freely walking on the cave floor.
The Ghaub cave is currently used for touristic purposes, receiving visitors regularly. However, apart from the rudimentary stone stairway at the gate entrance in the first conduit, no other man-made structures are present in the cave. The only altered area in the cave due to the tourism is a small chamber at the lower level, which is quite moist with mud. In this chamber, locals used to make mud animals that were left in some parts of the chamber, forming curious sets of mud figures (Fig. 8E). Fortunately, the cave sections where N. ghaubensis sp. nov. occurs are difficult to access, which minimizes the threat to this species by visiting tourists. The region's climate is considered as hot semi-arid (BSh), according to the updated Koppen-Geiger classification (Beck et al. 2018). According to historical data from 1982 to 2012, the average annual rainfall in the area ranges from 500 to 600 mm, with the wet season occurring from November to March (Merkel 2019). The cave is located in the "Angolan Mopane Woodlands" ecoregion (Dinerstein et al. 2017). The surface environment surrounding the cave is protected and located in a reserve that hosts tourists also for photographic safaris. The Otavi mountains are, in general, well preserved, with the land cover dominated by natural forests (Fig. 8F). Description. Maximum length: male, 6 mm. Colorless (Fig. 9A, B). Dorsal surface smooth with fan-shaped scale-setae (Fig. 10C); one line of noduli laterales per side on pereonites, at certain distance from lateral margin, b/c and d/c co-ordinates as in Fig. 10A; some gland pores on pereonites 1 to 7 (Fig. 10C). Cephalon (Figs 9B, 10B) with no suprantennal line; eyes reduced to small dot of pigment (Fig. 9B), absent in some specimens. Pleon slightly narrower than pereon, pleonites 3-5 epimera with triangular posterior points. Telson (Fig. 10D) triangular with slightly concave sides and rounded apex. Antennula (Fig. 10E) with three articles, second article shortest, distal article with ca. seven aesthetascs. Antenna (Fig. 10F) reaching pereonite 2 when extended backward; fifth article of peduncle as long as flagellum;  flagellum with two articles, second article about 3 times as long as first. Mandibles (Fig. 10G, H) with molar penicil consisting of several setae arising from common stem; left mandible with 2+1 penicils, right mandible with 1+1 penicils. Maxillula (Fig. 10I) outer branch with 4 + 5 teeth (two of them cleft); inner branch with two penicils. Maxilla (Fig. 10J) with bilobate apex, inner lobe distinctly smaller than outer lobe. Maxilliped (Fig. 10K) basis rectangular; palp basal article with two stout setae, distal article with apical tuft of setae; endite rectangular, apex with one long seta. Pereopod 1 (Fig. 11A) carpus with longitudinal grooming brush, inner claw of dactylus thin and shorter than outer one. Uropod (Fig. 10D) protopod surpassing distal margin of telson, lateral margin grooved with gland pores; endopod inserted slightly proximally, shorter than exopod.
Taxonomic remarks. Niambia namibiaensis sp. nov. is easily distinguishable from N. botswanaensis sp. nov. and N. ghaubensis sp. nov. in having some gland pores on the lateral margins of the pereonites, a rounded apex of telson, a brush of trifid setae on the male pereopods 1-4 merus and carpus, and a different shape of the male pleopod 1. It also differs from N. botswanaensis sp. nov. in having the dactylus of pereopods with a thinner inner claw. Ecological remarks. The Märchen cave and the Ghaub cave are both located in the Otavi Mountains, about 56 km apart. The Märchen cave presents a 12 m vertical shaft at the entrance with the maximum cave depth being 60 m. Farmers installed metal ladders to facilitate access to the cave (Fig. 12A-C). Specimens of N. namibiaensis sp. nov. were only found in one of the deepest portions of the cave, under rocks on the floor. This was also the only area with noticeable moisture content (Fig. 12D). Since the visit to the cave occurred in the dry season, most of the cave's substrates were extremely dry. In the humid part of the cave, where the isopods were found, other invertebrates were also observed, including potential predators such as the troglophilic Bothriuridae scorpion Lisposoma josehermana Lamoral, 1979 and spiders. The cave was not visited for research purposes during the rainy season and therefore it is not possible to determine if the distribution of this species within the cave is subject to seasonality. However, they may likely present a wider distribution during the wet periods since there are many signs of water drips in the cave. Although other caves were also sampled in the same outcrop, no specimens of N. namibiaensis sp. nov. were found. The external environment is well preserved (Fig. 12D) and even with the facilities to access the cave, there are no signs of human impact inside it. This species is not currently considered threatened.