Integrative revision of the giant pill-millipede genus Sphaeromimus from Madagascar, with the description of seven new species (Diplopoda, Sphaerotheriida, Arthrosphaeridae)

Abstract The Malagasy giant pill-millipede genus Sphaeromimus de Saussure & Zehntner, 1902 is revised. Seven new species, S. titanus sp. n., S. vatovavy sp. n., S. lavasoa sp. n., S. andohahela sp. n., S. ivohibe sp. n., S. saintelucei sp. n., and S. andrahomana sp. n. were discovered, in one case with the help of sequence data, in the rainforests of southeastern Madagascar. The species are described using light- and scanning electron microscopy. A key to all 10 species of the genus is presented. All but one (S. andohahela) of the newly discovered species are microendemics each occurring in isolated forest fragments. The mitochondrial COI barcoding gene was amplified and sequenced for 18 Sphaeromimus specimens, and a dataset containing COI sequences of 28 specimens representing all Sphaeromimus species (except S. vatovavy) was analyzed. All species are genetically monophyletic. Interspecific uncorrected genetic distances were moderate (4–10%) to high (18–25%), whereas intraspecific variation is low (0–3.5%). Sequence data allowed the correct identification of three colour morphs of S. musicus, as well as the identity of a cave specimen, which although aberrant in its morphology and colouration, was genetically identical to the holotype of S. andrahoma.


Introduction
Madagascar, the world's third largest island, is famous for its endemic and endangered fauna and flora (Myers et al. 2000, Vences et al. 2009). Some representatives of the millipedes, class Diplopoda, represent quite charismatic invertebrate endemics of Madagascar. These include the large-bodied, strikingly red-black colored socalled 'Fire-Millipedes' of the order Spirobolida (Wesener et al. 2009a, Wesener et al. 2011a, and the giant pill-millipedes, locally called 'Tainkintana' (=star droppings), reaching the size of a small orange or a tennis ball when rolled-up.
Despite their conspicuousness, it was only recently that the millipede biodiversity on Madagascar became better known. Mauriès (1994Mauriès ( , 1997 discovered and described the first African representatives of the order Chordeumatida from Madagascar, which belong to the same family Pygmaesomatidae as certain Indian endemics. Inventories of the 'classical' millipedes of the order Spirobolida led to the discovery of 13 new genera and 53 new species , Wesener and Enghoff 2009, Wesener et al. 2009b. Recently the occurrence of the order Polyzoniida on Madagascar was reviewed, and was found to be diverse and indigenous (Wesener 2014a), while representatives of the order Siphonophorida were discovered on Madagascar for the first time (Wesener in press) In giant pill-millipedes, all species known from Madagascar were redescribed Sierwald 2005, Wesener and. New discoveries included the first dwarfed member of the giant pill-millipede order, the genus Microsphaerotherium Wesener & VandenSpiegel, 2007, as well as a surge of species in the genus Zoosphaerium Pocock, 1895, which now includes 62 described species (Wesener 2009), including the largest Sphaerotheriida by far, e.g. Z. neptunus (Butler, 1872) reaching the size of a tennis ball or small orange when rolled-up.
The third Malagasy giant pill-millipede genus Sphaeromimus de Saussure & Zehntner, 1902, is an unusual representative of the order. One characteristic of the genus is the presence of well-developed stridulation organs, the male 'harp' and the female 'washboard', which carry more stridulation ribs than in any other member of the Sphaerotheriida. These stridulation organs are still not well understood, but may play a role during courtship (Wesener et al. 2011b). The first species, S. musicus was described in 1897 (de Saussure and Zehntner 1897), with no additional specimens found for more than 100 years. The unusual morphology of Sphaeromimus, quite distinct from the majority of Malagasy giant pill-millipedes belonging to the genus Zoosphaerium Pocock, 1895, let the experts to suggest that S. musicus might represent a "mislabelled or an introduced Indian sphaerotheriid" (Jeekel 1999). However, a century later, two additional species of Sphaeromimus were discovered in littoral rainforest fragments in southeastern Madagascar, and its type species could be redescribed based on numerous samples taken from the southern Malagasy spiny forest ecosystem during general biodiversity inventory programs (Wesener and Sierwald 2005). A phylogenetic analysis of the Sphaerotheriida based on morphological VandenSpiegel 2009, Wesener 2014b) as well as molecular characters (Wesener et al. 2010) confirmed the sister-group relationship of Sphaeromimus to the Indian genus Arthrosphaera Pocock, 1895, the first time such a Madagascar-India relationship was discovered in soil arthropods. Sphaeromimus is more closely related to the Indian genus Arthrosphaera than to the other Malagasy giant pill-millipede genera Zoosphaerium and Microsphaerotherium, all of which belong to the family Arthrosphaeridae (Wesener 2014b).
An expedition to Madagascar conducted by TW in 2007, as well as sorting through different natural history collections, led to the discovery of 12 additional Sphaeromimus populations representing seven undescribed species of Sphaeromimus, all from humid forests in southeastern Madagascar. Many of the newly discovered species were only found in tiny rainforest vestiges/fragments, and one specimen was discovered in a cave located in the southern dry spiny forest ecosystem. While all known Sphaeromimus show a 'normal' size of 18-35 mm, one of the undescribed species shows gigantism. These findings highlight how little we still know about the biodiversity of one of the most striking invertebrate endemics on Madagascar.

Specimen collecting and conservation
Sphaeromimus specimens were collected by hand. Three of the seven newly discovered species were found in natural history collections: one historic and two obtained during general arthropod inventory programs on Madagascar. Specimens of the other four species were collected from eight localities during an expedition by TW and Kai Schütte (University of Hamburg). Between 12 -20 hours were spent searching at each locality. Rarely were the Sphaeromimus individuals encountered in high numbers (spiny forest, Sainte Luce S9, Grande Lavasoa) and usually several hours of search were necessary to find an area where 3-5 specimens could be collected. The isolated occurrence patterns of Sphaeromimus species might be the main reason why no Sphaeromimus specimens were collected during general inventory programs, which targeted the same localities as we did in 2007. Legs were removed from several specimens and placed in 95% ethanol for DNA analysis, while the rest of the specimens was preserved in 80% ethanol, which was changed twice. A few months later, some of the specimens were transferred to 95% ethanol to facilitate future DNA work on the specimens. Table 1: Sphaeromimus samples, Genbank code, and depository. Genbank numbers marked by an asterisk (*) were published in a previous study (Wesener et al. 2010).

Illustrations
Important structures of the Sphaeromimus specimens were drawn using a camera lucida mounted on an Olympus SZX12 stereo-microscope. Pencil drawings were later transferred to ink with pigma micron pens. For scanning electron microscopy, samples were dehydrated via an ethanol series (90%, 95%, 2× 100%), dried over night, and mounted on aluminium stubs before being sputter coated with gold. SEM images were taken using a Zeiss Leo EVO SEM (FMNH) and a Hitachi S-2460 SEM (ZFMK). All images were later modified using Adobe Photoshop CS2 and assembled into plates using Adobe Illustrator CS2.

DNA extraction, sequencing
DNA was extracted from 18 specimens: 12 of them preserved in 95% ethanol, the remaining ones in 75% ethanol. The HCO/LCO primer pair (Folmer et al. 1994) was used to sequence a 674 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, does not allow a reconstruction of the phylogeny of the Sphaeromimus 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 obtained sequences were checked via Blast searches (Altschul et al. 1997), no contaminations were discovered. The sequences were aligned by hand in BioEdit (Hall 1999) with those obtained during a previous study (Wesener et al. 2010) from other Sphaeromimus specimens, using as outgroup taxa a specimen of the basal family Procyliosomatidae (Wesener and VandenSpiegel 2009), as well as two species of the other Malagasy genus Zoosphaerium, including the type Z. neptunus, and a member of the closely related Indian genus Arthrosphaera. All newly sequenced Sphaeromimus sequences were uploaded to Genbank (Accession #: KJ13244-KJ13261, see Table 1).

DNA analysis
To find the best substitution model, modeltest implemented in MEGA 5.05 (Tamura et al. 2011) was utilized. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 567 positions in the final dataset. The lowest Bayesian Information Criterion score of 8149.1 was obtained by the GTR model plus invariant sites and gamma distribution to be best fitting (FreqA = 0.2694, FreqC = 0.2286, FreqT = 0.3304, FreqG = 0.1716, Invariant sites = 0.535, gamma shape = 1.29762). Maximum likelihood analyses were conducted in MEGA5 (Tamura et al. 2011). The bootstrap consensus tree (Fig. 20) from 1000 replicates (Felsenstein 1985) is taken to represent the evolutionary history of the analyzed taxa. All positions containing gaps and missing data were eliminated. There were a total of 570 positions in the final dataset. Mean uncorrected pairwise distances between terminals (transformed into percentages) were determined using MEGA5 (Tamura et al. 2011 (Wesener and Vanden-Spiegel 2009;Wesener 2014b), and molecular characters (Wesener et al. 2010). A short re-diagnosis is presented so that fewer characters need to be mentioned in the species descriptions.
New diagnosis. Genus of small to medium-sized (15-45 mm length) Arthrosphaeridae. Colour variable, rarely pink or with a reddish-black pattern (Fig. 1A), but usually black to brown (Fig. 1B). Head with short antennae consisting of well-rounded antennomeres lacking cuticular scales. Antennomere 6 massive, only antennomere carrying sensilla basiconica. Apical disc with numerous apical cones (>>20), number of cones sexual dimorphic, males with twice or even three times as many cones as females. Eyes consisting of 55-95 ocelli. Mandible with six or seven pectinate lamellae and a 3-combed internal tooth. Gnathochilarium typical of the order, rudimentary lateral palpi carrying three or four sensory cones. Tergites with a smooth surface, in some species polished. Legs short and broad, tarsus usually 2.5-4 times longer than wide. Leg pair 1 and 2 lacking an apical spine. Femur often with a well-developed toothed ridge. Coxae with a more or less well-developed sharp process carrying small triangular spines. Anal shield usually well-rounded, underside carrying a single, short, black locking carina located closely to the margin.
Male gonopore typical of Arthrosphaeridae, covered by a simple sclerotized plate. Anterior telopod consisting of syncoxite plus four telopoditomeres. First telopoditomere enlarged, carrying the male stridulation organ (harp) consisting of 3-6 stridulation ribs, number of ribs independent of the size of the male, species specific. Second telopoditomere posteriorly with a process protruding up to the start of telopoditomere 4. Third telopoditomere short, sometimes with a spine juxtaposed to process of telopoditomere 2. Telopoditomere 4 larger than 3 and 4 combined, conical, basally with a field of sclerotized spots juxtaposed to process of telopoditomere 2, apically with a large, triangular lobe-like spine, usually three smaller spines distributed across the joint.
Posterior telopods always consisting of syncoxite with inner horns and inner lobes and pair of telopodites each consisting of three telopoditomeres forming chelae. First telopoditomere unremarkable, second telopoditomere forming the immovable finger of the chela. Immovable finger basally wide, apically tapering, always curved toward telopoditomere 3 (movable finger). Immovable finger as long as, or in some species overlapping movable finger. Membrane of telopoditomere 2 often extended into lobe, an anterior side juxtaposed to telopoditomere 3 with a dense field of sclerotized spots. Telopoditomere 3 curved or straight, relatively slender, margin juxtaposed to immovable finger with 20-40 small black crenulated teeth, three or four spines, and one, rarely two (S. ivohibe sp. n.) large membranous lobes.
Female vulva atypical for Arthrosphaeridae, with external and inner plate standing below operculum, not extending mesally. Operculum large and well-rounded, protruding at least up to basal half of prefemur. Female subanal plate greatly enlarged, in some species almost divided into two plates. Subanal plate carrying a washboard with 3-8 stridulation ribs on each side, number of ribs depending on female body size.
Colouration of tergites dark brown with black posterior margin. Paratergite impressions and groove of thoracic shield orange. Legs, antennae and pleurites orange, head and collum dark brownish-black, eyes green.
Etymology. 'titanus', adjective, referring to the large size of the species. Distribution. Only known from the eastern lowland rainforest of Manombo, which is now isolated by vast areas of pseudosteppe from all other remaining rainforests.
Diagnosis. Five stridulation ribs on the male harp, a character only shared with the spiny forest species S. musicus. Shape of female operculum unique, apically recessed. Endotergum with two rows of deeply impressed cuticular patterns and two dense, irregular rows of marginal bristles that protrude above tergite margin.
Endotergum inner section with numerous short triangular spines and long setae (Fig. 5B). Between ridge and inner area two rows of strongly impressed, circular cuticular impressions. Externally two dense but irregular rows of marginal bristles (Fig. 5B). Bristles long, protruding beyond tergite margin.
Female sexual characters: Coxa process on leg 2 well-developed. Vulva massive. Operculum apically emarginate, protruding up to basal half of prefemur (Fig. 9F). Subanal partly reduced, with shallow invagination at apical margin. Washboard with 10 stridulation ribs on each side.
Distribution. Only known from the eastern lowland rainforest of Tsianovoha. Satellite images do not show much remaining natural vegetation in the area.    Other material. ~ 50 ♂, ♀, juv., same data as holotype, sent as voucher specimens to the University of Antananarivo.

Sphaeromimus lavasoa
Diagnosis. Small shiny-black Sphaeromimus with brown collum and head. Male harp with four stridulation ribs. Midbody legs with weakly-developed coxal process.
Colouration of tergites black. Collum and head light brown (Fig. 1B). Legs and antennae dark greenish-brown.
Collum glabrous except 3 or 4 short isolated setae on the surface and few at margin. Thoracic shield smooth and glabrous, few setae in grooves. Tergites 3-12 smooth, paratergite tips of midbody tergites only weakly projecting posteriorly (Fig. 1B).
Anal shield massive, well-rounded, lacking pubescent area. Endotergum inner section with numerous short triangular spines and very few setae (Fig. 12A). Between ridge and inner area two rows of weakly impressed, circular cuticular impressions. Externally two irregular, dense rows of marginal bristles (Fig. 12A). Bristles short, ending well before tergite margin.
Female sexual characters: Second leg pair with well-developed coxal lobe.Vulva massive. Operculum well-rounded, protruding up to basal half of prefemur (Fig. 11F). Subanal shield almost divided into two, with strong invagination at apical margin (Fig.  11G). Washboard with three stridulation ribs on each side (Fig. 11G).
Etymology. 'Lavasoa', noun in apposition, after the Lavasoa (also called Ambatotsirongorongo) mountain, to which this species is endemic.
Distribution. Endemic to the Lavasoa Mountain, where it could be recorded from two of the three remaining fragments. The species was common in the largest fragment of Grande Lavasoa, but only a single female could be collected at Petit Lavasoa. Diagnosis. Small matte-black pill millipede with a dark brown head and collum and light brown appendages. Male harp with three stridulation ribs.
Colouration of tergites black, matte not shiny. Collum and head dark brown (Fig.  20). Depressions of paratergites as well as legs and antennae brownish.
Anal shield massive, well-rounded, lacking pubescent area. Endotergum inner section with numerous short triangular spines and very few setae (Fig. 12B). Between ridge and inner area two rows of weakly impressed, circular cuticular impressions. Externally two irregular rows of marginal bristles (Fig. 12B-D). Bristles short, barely protruding up to tergite margin.
Female sexual characters: Second leg pair with well-developed coxal lobe. Vulva massive. Operculum well-rounded, protruding above basal half of prefemur (Fig.  14E). Subanal shield almost divided into two, with strong invagination at apical margin. Washboard with three stridulation ribs on each side.
Intraspecific variation: The endotergum differs slightly in the development of the cuticular impressions between the specimens from Isaka-Ivondro (Fig. 12B), Malio (Fig. 12C) and Manantantely (Fig. 12D). The specimens from the three localities also differ slightly in their colour pattern: In specimens from Manantantely and Isaka-Ivondro, the head and collum are dark brown, while those from Malio have a brown thoracic shield too.
Genetic distances in the COI gene between the three populations are 2.9-4%, while even the two individuals from Malio show a variation at the population level of 3.6%. Future studies involving more localities and specimens should investigate whether or not gene flow occurs between the different populations of S. andohahela.  Etymology. 'andohahela', noun in apposition, after the type locality, the rainforests of the national park Andohahela.
Distribution. Widespread in the lowland and montane rainforests of the northern Anosy and Vohimena mountain chains. Diagnosis. Small shining black pill millipede with orange-reddish appendages. Of all currently known Sphaeromimus with highest number of stridulation ribs, 6, on male harp. Posterior telopods unique, with two large membranous lobes.
Colouration of tergites shining black. Paratergite impressions and groove of thoracic shield dark greenish. Legs, antennae and pleurites orange-red, eyes green.
Anal shield well-rounded, lacking pubescent area. Endotergum inner section with numerous short triangular spines and long setae (Fig. 16A). Between ridge and inner area two rows of weakly impressed, circular cuticular impressions. Externally single, sparse row of marginal bristles (Fig. 16A). Bristles short, not protruding up to tergite margin.
Distribution. Only known from the type locality. Diagnosis. S. saintelucei sp. n. shares its small size (<20 mm), total absence of a coxal lobe at midbody legs in combination with slender posterior telopods of which the apex of the immovable finger is strongly curved only with S. inexpectatus Wesener & Sierwald, 2005. S. saintelucei differs from the latter in the dull brown colour (shiny pink in S. inexpectatus), and the anterior telopods. Both species differ genetically at 4-4.8% of their COI gene.
Female unknown. Etymology. 'saintelucei', adjective, after the type locality, and only area of occurrence, the littoral rainforest of Sainte Luce.
Distribution. Only known from the only remaining southern lowland forest on basaltic soil, the tiny fragment S8 of Sainte Luce. In the nearby fragments on sandy soil, S. splendidus occurs. Diagnosis. Small matte-black pill millipede with a dark brown head and collum. Similar to S. andohahela but differs from the latter in weakly developed cuticular patterns and presence of numerous small pits on movable finger of posterior telopod. Genetical distance of the COI gene between both species is 10-11.4%.
Anal shield well-rounded, lacking pubescent area. Endotergum inner section with few short triangular spines and long setae (Fig.  16C). Between ridge and inner area two rows of weakly impressed, circular cuticular impressions. Externally two dense rows of marginal bristles (Fig. 16C). Bristles long, protruding above tergite margin.
Female unknown. Etymology: 'andrahomana', noun in apposition, after the famous cave close to the type locality, the Grotte d'Andrahomana.
Distribution. Relic occurrence in the Grotte D'Andrahomana. The single individual found close to the village Ankapaky might be an indication of a more widespread occurrence in the little explored Vohisandria and Amboalaingo hills N. of Ankapaky and S. of Ranopiso.
Discussion. Intraspecific variations: The cave specimen shows a distinct colour pattern: tergites very light brown with dark brown posterior margins (Fig. 1C), head and collum light brown, legs and antenna, at least apically red. The endotergum shows fewer spines and hair in the cave specimen (Fig. 16D) when compared to the holotype (Fig. 16C). Furthermore, the leg of the cave specimen is distinctively slenderer at its basal joints (Fig. 18B), the femur being 2.2 (holotype 1.6), the tarsus 3 (holotype 3) times longer than wide. Despite their large morphological difference show both specimens the same COI haplotype.

Updated distribution of Sphaeromimus:
Despite the description of seven new species and numerous additional localities Sphaeromimus is still restricted to southeastern Madagascar (Fig. 19). Most species occur in the rainforest and littoral rainforest, with only the widespread S. musicus occurring in the spiny forest ecosystem. Sphaeromimus specimens could be discovered in every single explored southeastern Malagasy rainforest (Fig. 19), always in sympatry with species of the other Malagasy genus of giant pill-millipedes, Zoosphaerium (see Wesener 2009).

Genetic distances between Sphaeromimus species
The analysis of the barcoding fragment of the COI gene provided a good resolution at the species level; all Sphaeromimus species are monophyletic and form well-supported terminals (Fig. 20). Genetic distances between the different Sphaeromimus species is 4.0% (S. inexpectatus and S. saintelucei) but mostly between 8-20% and up to 25.3% (S. musicus and S. splendidus). Based on their mitochondrial DNA, the species of the genus can be separated into two groups, albeit without any statistical support: (1) the extreme southeastern humid forest clade, and (2) a clade incorporating the spiny forest S. musicus together with the slightly more northern rainforest species from Ivohibe, Vevembe and Manombo (Fig. 20).
In the latter clade, all species show high genetic distances of 16-21% to one another and no sub-grouping receives any statistical support. More structure can be observed in the extreme southeastern clade. S. inexpectatus from the littoral rainforest of Mandena and the rainforest of Enato forms a well-supported monophyly with S. saintelucei from the nearby littoral rainforest fragment on basaltic soil at Sainte Luce S8. This monophyly is juxtaposed to a clade comprising S. andrahomana, S. lavasoa, S. splendidus, and S. andohahela. Within this clade, S. andrahomana, the southern-most taxon, is in the basal-most position differing by more than 10% of its base pairs from any other Sphaeromimus species (Fig. 20). S. andohahela from the Andohahela and Vohimena mountains is sister to a clade comprising the well-supported (69%) S. splendidus from the littoral rainforest sandy soil fragment S9 at Sainte Luce and S. lavasoa from the southern isolated Lavasoa Mountain.

Incorporating COI barcode data into the taxonomy of Sphaeromimus
Genetic distances between the species of Sphaeromimus are high, hinting at an old age of the speciation events shaping the current species of the genus (see also Wesener et al. 2010). This presumably old age is further highlighted by the fact that the deeper branches receive very little statistical support (Fig. 20). The COI gene probably already lost its resolution because too many reverse substitutions occurred.
Nevertheless, the COI gene is a powerful taxonomic tool, greatly improving our systematic understanding and has led to the description of new species in the family Zephroniidae from Asia (Wongthamwanich et al. 2012, Golovatch et al. 2012, and here also in the Malagasy genus Sphaeromimus. The incorporation of the COI barcoding gene allows a better separation of the small black taxa in the extreme southeastern clade which are more difficult to distinguish (Fig. 20). The COI data further led to the direct discovery of a pseudo-cryptic species, S. saintelucei. The few obtained Sphaeromimus specimens from the heavily degraded littoral rainforest fragment S8 at Sainte Luce (Fig. 19) were first mistaken for juveniles of S. splendidus. Only the very high genetic distances observed prompted a more close morphological study, which confirmed a closer morphological similarity with S. inexpectatus, matching the results from the analysis of the COI gene (Fig. 20). Additionally, the different colour morphs of S. musicus (Fig. 1A) could be correctly determined as just that, based on their identical COI sequences. The cave specimen of S. andrahomana, quite unusual in its colouration (Fig. 1C) and also morphology ( Fig.  18B) would have been described as a separate species if not for the 0% difference in its COI sequence with those of the holotype of S. andrahomana (Fig. 20).
The interesting relationships and biogeographic patterns among the species of Sphaeromimus, with species like S. splendidus and S. saintelucei occurring in close proximity to one another (Fig. 19), but showing great genetic distances (21.7%) and no close relationship (Fig. 20), are further hints to the interesting biogeographic mechanisms shaping the current distribution of Sphaeromimus species in southeastern Madagascar. These patterns should be studied further using more molecular markers. Ubick (CAS), who loaned out the specimens of S. titanus. Kai Schütte (Zoological