Ecotone shifts in southern Madagascar: first barcoding data and six new species of the endemic millipede genus Riotintobolus (Spirobolida, Pachybolidae)

Abstract Six new species of the Spirobolida millipede genus Riotintobolus Wesener, 2009, are described from the spiny forest in southern Madagascar utilising genetic barcoding, drawings and scanning electron microscopy: Riotintobolus tsimelahysp. nov., R. mangatsiakasp. nov., R. lavanonosp. nov., R. bovinussp. nov., R. antafokysp. nov. and R. makayisp. nov. One other Riotintobolus population from the spiny forest might represent an additional species based on genetic data, but it cannot be described as no male specimens were collected. At present, the genus Riotintobolus Wesener, 2009 has eight species from the spiny forest and two species from the littoral rainforest. A determination key to all ten species of the genus is provided. Molecular data reveal that the two critically endangered species from the humid littoral rainforest are not closely related to one another, but have their closest relative in the dry spiny forest ecosystem. Riotintobolus mandenensis Wesener, 2009, only known from the southern littoral rainforest of Mandena is related to R. tsimelahysp. nov. from the nearby spiny forest at Tsimelahy with a p-distance of 11%, while R. minutus Wesener, 2009 from the littoral forest of Sainte Luce is more distant to all other Riotintobolus species, but more closely related to R. bovinussp. nov. from the southwestern forest of the Makay.


Introduction
Madagascar is one of the ten hottest biodiversity hotspots (Myers et al 2000). The long isolation of Madagascar from other land masses (Krause 2003;Ali and Aitchison 2008) makes the island a great model to study the speciation mechanisms in numerous of its endemic animal groups (Vences et al. 2009). The evolutionary mechanisms among the rise of such a stunning diversity of a high number of animal groups on Madagascar are still little understood, as most studies focus on the vertebrate fauna of the island (e.g. Pearson and Raxworthy 2009). Invertebrates might be better suited for studying biogeographical patterns, as they can survive in smaller habitat fragments during climatically unsuitable times (Brühl 1997;Uys et al. 2009;Yeates et al. 2009).
Among the endemic mega-invertebrate fauna on Madagascar are the so-called fire millipedes of the order Spirobolida with their striking red/black coloration (Wesener et al. 2009a(Wesener et al. , b, 2011a, and the large giant pill-millipedes of the order Sphaerotheriida with stridulation organs in both sexes (Wesener and VandenSpiegel 2009;Wesener et al. 2011b), reaching the size of a small orange when rolled-up (Enghoff 2003). For both groups, numerous species and genera could recently be described from Madagascar (Wesener et al. , 2011a(Wesener et al. , 2014Wesener 2009aWesener , b, 2011Wesener and Enghoff 2009). Especially diverse are the Spirobolida millipedes on Madagascar; previously only known from two genera, 14 new genera could be described since 2008, with numerous species being microendemic and even listed on the IUCN Red List as critically endangered (e.g. Rudolf and Wesener 2017a, b). One of these genera is Riotintobolus Wesener, 2009, aptly named after the mining company Rio Tinto, which is currently active in the only known southern littoral rainforests (Vincelette et al. 2007) where two of the critically endangered species of the genus live (Rudolf and Wesener 2017c, d). The other two species of the genus are known from the desert-like spiny forest ecosystem (Du Puy and Moat 1996;Moat and Smith 2007) in southern Madagascar and seem to have a more widespread distribution. Riotintobolus is therefore one of the few genera of Malagasy Spirobolida whose species, distributed in littoral rainforests and dry spiny forests, underwent one or several so-called ecotone shifts (Wirta et al. 2008;Wesener et al. 2011a).
An expedition to Madagascar conducted by TW in 2007, as well as sorting through different museum collections, led to the discovery of six additional Riotintobolus species, all from the spiny and gallery forests in the desert-like south of Madagascar.

CAS
California

Illustrations
Dissecting and camera lucida drawings were done under an Olympus SZX12 stereomicroscope. For scanning electron microscopy, the samples were dehydrated via an ethanol chain, mounted on stubs and dried overnight. The stub was sputter-coated with 100 nm of gold in a Hummer VI (Anatech, USA) sputtering system. Images were obtained using a Hitachi S-2460 SEM. Multi-layer photographs were taken with a Leica Z6 Imaging-System based at the ZFMK. Stacked images were put together using the software Auto-Montage (Syncroscopy). All images were later modified using Adobe Photoshop version CS2 and assembled into plates using Adobe Illustrator version CS2.

DNA extraction and sequencing
DNA was extracted from 14 specimens (see Table 1) of Riotintobolus: ten of them preserved in 95% ethanol, the remaining in 75% ethanol. The HCO/LCO primer pair (Folmer et al. 1994) was used to sequence a 652 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. DNA extraction, PCR, purification, and sequencing protocols were identical to those used in a previous study (Wesener et al. 2010). While the COI gene, being a mitochondrial gene as well as containing little resolution at deeper evolutionary splits, is limited in the resolution of a reconstructed phylogeny of the Riotintobolus species, we aimed at finding a unique identifier allowing us to study and illustrate the genetic distances between the different species of the genus. All sequences obtained were checked via Blast searches (Altschul et al. 1997), no contaminations were discovered. The sequences were aligned by hand in BioEdit (Hall 1999) together with those obtained during the only other molecular study on Malagasy Spirobolida (Wesener et al. 2011a), using as outgroup taxa specimens of the genera Spiromimus DeSaussure & Zehntner, 1901 and Aphistogoniulus Silvestri, 1897 as the near outgroup, and a sequence of the species Madabolus maximus Wesener & Enghoff, 2008 of the tribe Pachybolini as the far outgroup. All newly sequenced Riotintobolus sequences were uploaded to Gen-Bank (Accession #: MT603148-MT603161, see Table 1).

DNA analysis
To find the best substitution model, modeltest implemented in MEGA 6 (Tamura et al. 2013) was utilised. Codon positions included were 1st+2nd+3rd. All positions containing gaps and missing data were eliminated. There was a total of 652 positions in the final dataset. The lowest Bayesian Information Criterion score of 10760 was obtained by the Tamura-Nei model plus gamma distribution to be best fitting (FreqA = 0.2848, FreqC = 0.1882, FreqT = 0.3572, FreqG = 0.17, gamma shape = 0.4526). Maximum Likelihood analyses were conducted in MEGA6 (Tamura et al. 2013). The bootstrap consensus tree ( Fig. 1) from 1000 replicates (Felsenstein 1985) is taken to represent the evolutionary history of the analysed taxa. The tree with the highest log likelihood (-5174.8354) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.4535)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 19 nucleotide sequences. Codon positions included were 1st+2nd+3rd. All positions with less than 5% site coverage were eliminated. That is, fewer than 95% alignment gaps, missing data, and ambiguous bases were allowed at any position. There was a total of 652 positions in the final dataset. Evolutionary analyses were conducted in MEGA 6 (Tamura et al. 2013). Genetic distances were also analysed in MEGA 6. The analysis involved 19 nucleotide sequences. Codon positions included were 1st+2nd+3rd. All ambiguous positions were removed for each sequence pair. Results are shown in the supplemental material (Suppl. material 1).
Determination key to the species of Riotintobolus Etymology. Tsimelahy, after the type locality ( Fig. 2), spiny forests next to the Tsimelahy River, Andohahela National Parc. Noun in apposition.
Diagnosis. Riotintobolus tsimelahy sp. nov. shares the absence of a projecting epiproct on the telson only with R. anomalus, R. antafoky sp. nov., R. bovinus sp. nov., R. mangatsiaka sp. nov. and R. lavanono sp. nov. The posterior telopod featuring two slender, sharp projections is only shared with R. bovinus sp. nov., R. mangatsiaka sp. nov. and R. lavanono sp. nov. A posterior gonopod separated into three parts is only shared with R. mangatsiaka sp. nov. and R. lavanono sp. nov., whose habitus and gonopods look very similar to those of R. tsimelahy sp. nov. Both species differ in details of the tip of the posterior gonopod and in the colour of their antennae and legs, which are red in R. tsimelahy sp. nov. and dark grey in R. lavanono sp. nov. R. tsimelahy sp. nov. differs from R. mangatsiaka sp. nov. in the presence of two lateral processes on the posterior gonopod. All three species differ by >11% uncorrected p-distance in the COI barcoding gene.
Description. Measurements: Telson not included in counts of segments. Male holotype with 50+0 segments, ca. 44 mm long, 4.3 mm wide. Largest females of type series with 49 or 50+0 segments, up to 50 mm long, 5.3 mm wide.
Colour (in living specimens): Body rings grey, appendages red. Head, paraprocts and posterior margins of body segments darker grey to black. Ozopore openings highlighted by black spot (Fig. 3A, C). Labrum with standard three irregular teeth and a single row of 10-12 stout marginal setae. Clypeus with two setiferous foveolae on each side (Fig. 3B). Antennae long, protruding back to segment 5. Length of antennomeres: 1<2>3=4=5=6. Second antennomere slenderer but twice as long as first. Terminal antennomere with four large sensory cones located together inside a membranous area.
Gnathochilarium: lamellae linguales each with two standard setae located behind one another. Stipites each with three apical setae. Palpi of similar size. Endochilarium not dissected.
Mandible not dissected. Collum: smooth, laterally not protruding as far as ring 2 (Fig. 3A). Body rings: ozopores starting at segment 6, marked by a black spot. Located on suture between meso-and metazonite. Rings with smooth, but irregular coriaceous surface, ventrally on metazona with transverse ridges.
Legs: leg 1 with a large cylindrical coxa, twice as long as other podomeres. Tarsus with three pairs of ventral spines and an apical spine beyond claw. Leg 2 with an elongated coxa. Tarsus with three pairs of ventral spines and a short apical spine. Midbody legs with a rectangular coxa, as long as other podomeres. Each podomere ventrally with a single or a pair of apical setae, tarsus with a single apical and three pairs of ventral spines. Length of midbody legs ca. 1.2 times body diameter in males.
Female sexual characters. No tarsal pads, antennae shorter than male, only protruding back to ring 2. Vulvae not dissected.
Posterior gonopods consisting of three parts, separated by sutures or articulations: a basal coxite with a slender coxite projection and a slightly shorter telopodite, efferent duct discharging laterally (Fig. 3E, F). Process of coxite and telopodite standing in same axis (Fig. 3E, F). Pair of posterior gonopods located parallel to each other, connected by a small, sclerotised and visible sternite. Basal part of coxite wide, mesally with a large triangular sclerite located on lower level than remaining part. Coxite elongated. Efferent duct running at mesal margin of coxite ( Fig. 3E) before curving to the lateral margin at beginning of telopodite (Fig. 3E). Telopodite as wide as but much shorter than coxite, standing in same axis (Fig. 3F), apically membranous, with one triangular apical process and two slender lateral processes (Fig. 3E, F). Lateral processes straight, running almost parallel to one another, slender and sclerotised, efferent duct seems to be ending at base of lateral process (Fig. 3E). Base of lateral process with a short, membranous-white projection (Fig. 3E).
Intraspecific variation. The number of segments varies, even within one population, between 47 and 51. The population from the Forêt de Mahavelo (Fig. 1) differs by 1.5% uncorrected p-distance of the COI gene to those from the type locality, Tsimelahy (Suppl. material 1). One small male from the type series (ZFMK MYR9949) has incompletely developed posterior gonopods, either a sign of healed damage (there are black spots on it), or maybe not fully developed (FiIg. 3G, H).
Live observations. R. tsimelahy sp. nov. could be found in great numbers in the early morning (7-9 a.m.) on the forest floor of the spiny bush. The otherwise dry spiny bush was still quite wet because of dew. No juveniles were observed. Contrary to other Riotintobolus species, such as R. mandenensis and R. minutus, R. tsimelahy sp. nov. did not remain stiff like a stick when disturbed, but rolled-up into a spiral, a common defence behaviour for juliform millipedes. Etymology. Mangatsiaka, after the type locality (Fig. 2), spiny forests next to a site called Mangatsiaka, Andohahela National Parc. Noun in apposition.

Riotintobolus mangatsiaka
Diagnosis. Riotintobolus mangatsiaka sp. nov. shares the absence of a projecting epiproct on the telson only with R. anomalus, R. antafoky sp. nov., R. bovinus sp. nov., R. tsimelahy sp. nov. and R. lavanono sp. nov. The posterior telopod featuring two slender, sharp projections is only shared with R. bovinus sp. nov., R. tsimelahy sp. nov. and R. lavanono sp. nov. A posterior gonopod separated into three parts is only shared with R. tsimelahy sp. nov. and R. lavanono sp. nov., whose habitus and gonopods look very similar to those of R. mangatsiaka sp. nov. Both species differ in details of the tip of the posterior gonopod and in the colour of their antennae and legs, which are red in R. mangatsiaka sp. nov. and dark grey in R. lavanono sp. nov. R. mangatsiaka sp. nov. differs from R. tsimelahy sp. nov. in the presence of just one lateral processes on the posterior gonopod. All three species differ by >11% uncorrected p-distance in the COI barcoding gene.
Colour (in living specimens): Body rings grey, appendages red. Head, paraprocts and posterior margins of body segments darker grey to black (Fig. 4A). Ozopore openings highlighted by black spot (Fig. 4A, B).
Legs: leg 1 with a large cylindrical coxa, twice as long as other podomeres. Tarsus with three pairs of ventral spines and an apical spine beyond claw. Leg 2 with an elongated coxa. Tarsus with three pairs of ventral spines and a short apical spine. Midbody legs with a rectangular coxa, as long as other podomeres. Each podomere ventrally with a single or a pair of apical setae, tarsus with a single apical spine and three pairs of ventral spines. Length of midbody legs ca. 1.2 times body diameter in males.
Female sexual characters. No tarsal pads, antennae shorter than male, only protruding back to ring 2. Female vulva simple, bivalve-like (Fig. 6B). Anterior plate smaller than posterior plate, opening with one row of setae on each plate, close to operculum.
Posterior gonopods consisting of three parts, separated by sutures or articulations (Fig. 4H): a basal coxite with a slender coxite projection and a slightly shorter telopo-dite, efferent duct discharging laterally (FIg. 4H, I). Process of coxite and telopodite standing in same axis (Fig. 4H). Pair of posterior gonopods located parallel to each other, connected by a small, sclerotised and visible sternite (Fig. 4H). Basal part of coxite wide, mesally with a large triangular sclerite located on lower level than remaining part (Fig. 4H). Coxite elongated. Efferent duct running at mesal margin of coxite (FIg. 4H, I) before curving to the lateral margin at beginning of telopodite (Fig. 4I). Telopodite as wide as but much shorter than coxite, standing in same axis (FIg. 4H, I), apically membranous, with two slender apical processes both diverging (FIg. 4H, I). Mesal process membranous and wider, lateral process longer, slenderer and sclerotised, efferent duct seems to be ending at base of lateral process (FIg. 4H, I). Base of lateral process with a short, membranous-white projection (Fig. 4I).
Intraspecific variation. The number of segments varies between 47 and 51. Live observations. R. mangatsiaka sp. nov. could be found in great numbers in the early morning (7-9 a.m.) on the forest floor of the spiny bush. The otherwise dry spiny bush was still quite wet because of dew. No juveniles were observed. Contrary to other Riotintobolus species, such as R. mandenensis and R. minutus, R. mangatsiaka sp. nov. did not remain stiff like a stick when disturbed, but rolled-up into a spiral (Fig. 4B), a common defence behaviour for juliform millipedes.
Diagnosis. Riotintobolus lavanono sp. nov. shares the absence of a projecting epiproct on the telson with R. anomalus, R. antafoky sp. nov., R. bovinus sp. nov., R. tsimelahy sp. nov. and R. mangatsiaka sp. nov., The posterior telopod featuring two slender, sharp projections is only shared with R. bovinus sp. nov., R. mangatsiaka sp. nov. and R. tsimelahy sp. nov. A posterior gonopod separated into three parts is only shared with R. mangatsiaka sp. nov. and R. tsimelahy sp. nov., whose habitus and gonopods look very similar to those of R. lavanono sp. nov. Both species differ in details of the tip of the posterior gonopod and in the colour of their antennae and legs, which are dark grey in R. lavanono sp. nov. and red in both R. mangatsiaka sp. nov. and R. tsimelahy sp. nov. All three species differ by >11% uncorrected p-distance in the COI barcoding gene.
Description. Measurements: male holotype with 47+0 segments, ca. 42 mm long, 4.2 mm wide. Largest females of type series with 46-48+0 segments, up to 58 mm long, 6.4 mm wide. Colour (in living specimens): Body rings and head grey, appendages black (Fig. 7A). Paraprocts and posterior margins of body segments darker grey to black (Fig. 7B). Ozopore openings highlighted by black spot.
Gnathochilarium: lamellae linguales each with two standard setae located behind one another. Stipites each with three apical setae. Endochilarium not dissected.
Collum: smooth, laterally not protruding as far as ring 2 (Fig. 7A). Body rings: ozopores starting at segment 6, marked by a black spot. Located on suture between meso-and metazonite. Rings with smooth, but irregular coriaceous surface, ventrally on metazona with transverse ridges.
Legs: leg 1 with a large cylindrical coxa, twice as long as other podomeres. Tarsus with three pairs of ventral spines and an apical spine beyond claw. Leg 2 with an elongated coxa. Tarsus with three pairs of ventral spines and a short apical spine. Midbody legs with a rectangular coxa, as long as other podomeres. Each podomere ventrally with a single or a pair of apical setae, tarsus with a single apical and four pairs of ventral spines. Length of midbody legs ca. 1.2 times body diameter in males.
Female sexual characters. No tarsal pads, antennae shorter than male, only protruding back to ring 2. Female vulva simple, bivalve-like.
Anterior gonopod sternite massive, elongated into a wide, well-rounded triangular lobe (Fig. 7C). Sternite in anterior view well-visible, without discernible apodemes, protruding almost as high as coxal processes. Coxite with a large, well-rounded mesal process. Telopodite with slender process arising mesally (Fig. 7C), process apically curved with a large triangular projection, tip well-rounded, slightly protruding above lateral margin of telopodite.
Posterior gonopods consisting of three parts, separated by sutures or articulations (Fig. 7D): a basal coxite with a slender coxite projection and a shorter telopodite, efferenct duct discharging laterally (Fig. 7E). Process of coxite and telopodite standing in same axis. Pair of posterior gonopods located parallel to each other, connected by a small, sclerotised and visible sternite. Basal part of coxite wide, mesally with a large triangular sclerite located on lower level than remaining part. Coxite elongated. Efferent duct running at mesal margin of coxite before curving to the lateral margin at beginning of telopodite (Fig. 7E). Telopodite half as wide and much shorter than coxite, standing in same axis, apically membranous, with two slender apical processes both diverging (Fig. 7D, E). Mesal process membranous and wider, lateral process bent 90 degrees laterally, longer, slenderer and sclerotised, efferent duct seems to be ending at base of lateral process (Fig. 7E). Base of lateral process with a short, membranous-white projection (Fig. 7E).
Intraspecific variation. Specimens of the same population differing between 45-47 in segment number. Females appear to be more brownish than the more greyish males.
Live observations. R. lavanono sp. nov. could be found in great numbers after a rainy day in the late afternoon (3-5 p.m.) in a small remnant of spiny bush and under dead Opuntia remains. The specimens were only encountered in an area of a few square meters in view of the coast. Contrary to other Riotintobolus species, such as R. mandenensis and R. minutus, R. lavanono sp. nov. did not remain stiff like a stick when disturbed, but rolled-up into a spiral.
Etymology: bovinus, after the gonopods which resemble the horns of a cow. Noun in apposition.
Diagnosis. Riotintobolus bovinus sp. nov. shares the absence of a projecting epiproct on the telson only with R. anomalus, R. antafoky sp. nov., R. tsimelahy sp. nov., R. mangatsiaka sp. nov. and R. lavanono sp. nov. The posterior telopod featuring two slender, sharp projections is only shared with R. tsimelahy sp. nov., R. mangatsiaka sp. nov. and R. lavanono sp. nov. R. bovinus sp. nov. differs from R. tsimelahy sp. nov., R. mangatsiaka sp. nov. and R. lavanono sp. nov. in a much smaller segment number and size, and strong differences in the posterior telopod, whose telopodite is uniquely shaped with two sharp processes running parallel to one another resembling a bull's horn. R. bovinus sp. nov. differs by more than 14% uncorrected p-distance in the COI barcoding gene from all other Riotintobolus species.
Description. Measurements: 41+0 segments. Ca. 25 mm long (broken), 2.4 mm wide. Colour (after 10 years in ethanol): Head and body rings grey, appendages red. Ventral site reddish. Posterior margins of body segments and whole margin of collum black. Anal valves black.
Gnathochilarium: lamellae linguales each with two standard setae located behind one another. Stipites each with three apical setae. Endochilarium not dissected.
Legs: leg 1 with a large cylindrical coxa, twice as long as other podomeres. Tarsus with three pairs of ventral spines and an apical spine beyond claw. Leg 2 with an elongated coxa and a strongly swollen prefemur. Tarsus with two pairs of ventral spines and a short apical spine. Midbody legs with a rectangular coxa, as long as other podomeres. Each podomere ventrally with a single or a pair of apical setae, tarsus with a tarsal pad, a single apical and two pairs of ventral spines. Length of midbody legs ca. 1.2 times body diameter in males.
Posterior gonopods consisting of two parts, separated by an articulation (Fig. 8F): a long coxite and a slightly shorter telopodite, efferent duct discharging apically (Fig. 8F, G). Process of coxite and telopodite standing in same axis (Fig. 8F). Pair of posterior gonopods located parallel to each other, connected by a small, sclerotised and visible sternite (Fig. 8F). Basal part of coxite wide, mesally with a large triangular sclerite located on lower level than remaining part (Fig. 8F). Coxite elongated. Efferent duct running at mesal margin of coxite (Fig. 8F, G). Telopodite as wide as but slightly shorter than coxite, standing in same axis (Fig. 8F, G), apically membranous, with two slender apical processes resembling a bull's horns (Fig. 8F, G). Mesal process wider and longer than lateral process. Efferent duct seems to be ending at base of mesal process (Fig. 8F, G).
Diagnosis. R. antafoky sp. nov. shares the flag-like membranous tip of the posterior gonopod with R. mandenensis, R. minutus, R. aridus, R. anomalus and R. makayi sp. nov. R. antafoky sp. nov. shares the absence of tarsal pads only with R. bovinus sp. nov., and the relatively simple tip of the posterior gonopod only with R. anomalus. R. antafoky sp. nov. differs from the sympatric R. anomalus in details of the posterior gonopod, the absence of a dorsal red stripe, the much longer antenna (protruding back to body ring 5), and the much smaller size (R. anomalus males 45 mm long, 4.3 mm wide, R. antafoky sp. nov. ca. 33 mm long, 3.2 mm wide).
Coloration: segments grey, with a dark grey posterior margin, ozopore highlighted by a black spot. Head, antennae and legs dark grey. Head: each eye with 34 ommatidia in six rows. Incisura lateralis open. Labrum with standard three irregular teeth and a single row of 10-12 stout marginal setae. Clypeus with two setiferous foveolae on each side. Antennae long, protruding back to segment 5. Terminal antennomere with four large sensory cones located together inside a membranous area. Antennomere 5 and 6 latero-apically with sensilla basiconica.
Gnathochilarium: lamellae linguales each with two standard setae located behind one another. Stipites each with three apical setae. Endochilarium not dissected.
Mandible: Stipes without projection, well rounded. Gnathal lobe not dissected. Collum: smooth, laterally not protruding as far as ring 2. Body rings: ozopores starting at segment 6, marked by a black spot. Located slightly before, but touching suture between meso-and metazonite. Rings with smooth, but irregular coriaceous surface, ventrally on metazona with transverse ridges.
Legs: leg 1 with a large cylindrical coxa, twice as long as other podomeres. Tarsus with three pairs of ventral spines and an apical spine beyond claw. Leg 2 with an elongated coxa and a strongly swollen prefemur. Tarsus with two pairs of ventral spines and a short apical spine. Midbody legs with a rectangular coxa, as long as other podomeres. Each podomere ventrally with a single or a pair of apical setae, tarsus without a tarsal pad, a single apical and two pairs of ventral spines. Length of midbody legs ca. 0.9 times body diameter in males.
Posterior gonopods consisting of two parts, separated by an articulation: a long coxite and a slightly shorter telopodite, efferent duct discharging apically (Fig. 9C, D). Process of coxite and telopodite standing in same axis (Fig. 9D). Pair of posterior gonopods located parallel to each other, connected by a small, sclerotised and visible sternite (Fig. 9D). Basal part of coxite wide, mesally with a triangular sclerite located on lower level than remaining part (Fig. 9C). Coxite elongated. Efferent duct running at mesal margin of coxite (Fig. 9C, D). Telopodite as wide as but slightly shorter than coxite, standing in same axis (Fig. 9D), apically with a membranous 'flag' (Fig. 9C, D). Laterally with sclerotised projection bending laterally and completely surrounded by membranous flag. (Fig. 9C, D). Efferent duct ending at base of process (Fig. 9C, D). Apex laterally with weakly developed finger-shaped process.
Remarks. this species lives in direct sympatry with the much larger R. anomalus.
Diagnosis. R. makayi sp. nov. shares the flag-like membranous tip of the posterior gonopod with R. mandenensis, R. minutus, R. aridus, R. anomalus and R. antafoky sp. nov. Riotintobolus makayi sp. nov. shares the wide dorsal stripe, presence of tarsal pads on at least male legs 3-7, and a projecting epiproct only with R. mandenensis, R. minutus, and R. aridus. R. makayi sp. nov. differs from R. mandenensis and R. minutus in the configuration of the flag of the posterior telopod, which consists only of a single fold, while it has two folds in the latter two species. The curved lateral process and the freely ending efferent duct are unique characters for R. makayi sp. nov.
Colour (after ten years in ethanol): Head except for light clypeus black, body rings black, appendages red (Fig. 10A, B). Anterior margin of collum light brown. Dorsally with two wide light brown-reddish stripes, divided by a black stripe, all stripes even crossing the epiproct. Anal valves black, margin light brown, hypoproct light brown (Fig. 10C).

Discussion
Genetic distances in the Barcoding gene of Riotintobolus species compared to other Diplopoda The interspecific distance of 11-16.4% between Riotintobolus species is comparable to those found in the only other Malagasy Spirobolida genus for which molecular data are available, Aphistogoniulus. Aphistogoniulus species are much more widespread and showing even higher interspecific distances (Wesener et al. 2009b(Wesener et al. , 2011a, being found all over Madagascar, while Riotintobolus is currently known only from the South. For other millipedes from Madagascar, interspecific distances are known for two genera of giant pill-millipedes (Sphaerotheriida), Sphaeromimus DeSaussure &Zehntner, 1902 andZoosphaerium Pocock, 1895. In Sphaeromimus, whose species show a distribution restricted to southern Madagascar comparable to those of Riotintobolus, interspecific distances vary mainly between 8.3-20.8% (Wesener et al. 2014;Moritz and Wesener 2017). In the widespread genus Zoosphaerium, only a handful of species were sequenced (Wesener et al. 2010;Sagorny and Wesener 2017;Wesener and Anilkumar 2020), with interspecific distances varying between 9.1-16.3%.

A hidden soil arthropod diversity in Madagascar's driest areas
Our discovery of six new species, especially of the occurrence of two unrelated species of Riotintobolus in direct sympatry and the presence of an additional candidate species, shows that Riotintobolus is an important component of the soil macrofauna in Madagascar's spiny forest ecosystem. Rakontondranary (Antananarivo). Our thanks go to Darrel Ubick (CAS), and Crystal Maier and Petra Sierwald (FMNH) for the arrangement of the loan of numerous specimens. The specimens from the Makay were collected by Jean Noel from the Association Mitsinjo and we thank him for the opportunity to study them. One fifth of the publication charge of this article was thankfully funded by the Open Access Fund of the Leibniz Association. Many thanks to the reviewers Henrik Enghoff and Sergei I. Golovatch, as well as to the subject editor Didier van den Spiegel for greatly improving the article.