Integrative description of two new species of Malagasy chirping giant pill-millipedes, genus Sphaeromimus (Diplopoda: Sphaerotheriida: Arthrosphaeridae)

The species-rich giant pill-millipedes (Sphaerotheriida) often represent a microendemic component of Madagascar’s mega-invertebrate fauna. Of the chirping genus Sphaeromimus de Saussure & Zehntner, 1902, ten species have been described. Here, we describe two new species of Sphaeromimus integratively, combining light microscopy, scanning electron microscopy, DNA barcoding and micro-CT technology for the fi rst time in a taxonomic description of a giant pillmillipede. S. kalambatritra sp. nov. and S. midongy sp. nov. are the fi rst giant pill-millipedes collected and described from the mountainous rainforests of Kalambatritra and Midongy. Both species show island gigantism compared to their congeners. Our analysis of the mitochondrial COI gene shows that the two species are related to one another with a moderate genetic distance (9.4%), while they are more closely related to an undetermined specimen from the forest of Vevembe (6.3% and 8.4%). They stand in a basal position with S. ivohibe Wesener, 2014 and S. musicus (de Saussure & Zehntner, 1897). The four aforementioned species share a high number of stridulation ribs on the male harp. Our microCT analysis provides a look into the head of S. kalambatritra sp. nov. and shows that non-destructive CT methods are a useful tool for studying the inner morphology of giant pill-millipedes.


Introduction
Madagascar, the world's 4 th largest island, lies in the Indian Ocean east of Africa and has a tropical climate (Moat & Smith 2007).While its center and west is mainly covered by dry savanna and dry forest, its east is covered by dense humid rainforests (Moat & Smith 2007).Madagascar is considered one of the world's hottest biodiversity hotspots (Myers et al. 2000;Ganzhorn et al. 2001) and provides shelter to a high diversity of endemic species.This high degree of endemism is probably due to its early isolation from Africa more than 150 Ma years ago and from India more than 80 Ma years ago (Wells 2003).Thus 99% of the amphibian species (Glaw & Verdes 2003) and 90% of all species of reptiles (Raxworthy 2003) and mammals (Goodman et al. 2003) in Madagascar are endemic.Since the arrival of humans ca 2000 years ago (Dewar & Wright 1993), the landscape of Madagascar has undergone tremendous changes resulting in only 9.9% of the primary vegetation remaining (Myers et al. 2000).A key thread to Madagascar's biodiversity is the fast rate of deforestation of habitat (Harper et al. 2007).Although several projects and action plans were established in the last decades to protect Madagascar's rich nature, deforestation and fragmentation still takes place at an increasing rate (Horning 2012;Waeber et al. 2016).Thus, from 1950 to 2000 the area covered by forests decreased by 40% (Green & Sussman 1990).
One of the highly endemic faunal groups of Madagascar are the millipedes, class Diplopoda (Enghoff 2003).Millipedes play an important role in terrestrial ecosystems as detritivores (Cárcamo et al. 2000) and were among the fi rst terrestrial animals, with fossils dating back to the middle Silurian (Shear & Edgecombe 2010).Sixteen extant orders of Diplopoda are known (Blanke & Wesener 2014), of which eight occur naturally on Madagascar (Shelley & Golovatch 2011), with two of them, Polyzoniida (Wesener 2014a) and Siphonophorida (Wesener 2014b), being only recently recorded from the island.Madagascar is host to some of the most spectacular millipedes: different genera of large-bodied pitchblack/bloodred so called "Fire-Millipedes" of the order Spirobolida (Wesener et al. 2009a(Wesener et al. , 2009b(Wesener et al. , 2011)), as well as the world's largest giant pill-millipedes, order Sphaerotheriida (see Wesener & Wägele 2008;Wesener 2009), showing island gigantism.All giant pill-millipedes of Madagascar belong to the family Arthrosphaeridae, otherwise only found in India (Wesener & VandenSpiegel 2009;Wesener et al. 2010) and are an example of the interesting biogeographical history of the Malagasy fauna.All species of the family are characterized by unique stridulation organs present in both sexes (see also Wesener 2014c), the male harp and the female washboard.On Madagascar, three genera with altogether 77 species of Arthrosphaeridae, all endemic, can be found (Wesener 2016): the species-rich genus Zoosphaerium Pocock, 1895, the dwarfed species of the genus Microsphaerotherium Wesener & VandenSpiegel, 2007 and the genus Sphaeromimus, whose members have especially well-developed stridulation organs (Wesener & Sierwald 2005).Until 2005 only known from a single specimen, Sphaeromimus was thought to be based on a mislabeled Indian specimen (Jeekel 1999), but then two new species and many specimens were described from SE Madagascar (Wesener & Sierwald 2005).Morphological (Wesener & VandenSpiegel 2009) and molecular (Wesener et al. 2010) studies suggest that Sphaeromimus is more closely related to the Indian genus Arthrosphaera Pocock, 1895 than to the other genera in Madagascar.Seven additional species were described integratively in 2014, including the fi rst gigantic species of the genus, reaching the size of a ping-pong ball (Wesener et al. 2014).All specimens found so far are from south and south-east Madagascar (Wesener 2016), and inhabit humid lowland, littoral, or montane rainforests, apart from a single species (S. musicus (Saussure & Zehntner, 1897)) which is widespread in the spiny forest ecosystem.
Here, we describe two new species of the genus Sphaeromimus, both of them "giants".The species come from previously unsampled regions, the quite remote (only approachable via a hike of >30 km) uniquely transitional forest of Kalambatritra (Irwin et al. 2001) and its nearest neighbour, the eastern montane rainforest of Midongy-Sud (Bora et al. 2007).We chose to describe both species integratively (Padial et al. 2010), using drawings and scanning-electron microscopy (Oatley et al. 1966) combined with genetic barcoding (Hebert et al. 2003;Hebert & Gregory 2005).For only the second time in Diplopoda taxonomy (Akkari et al. 2015), we also use micro-CT technology (Ritman 2004) to study and visualize internal structures non-invasively.

Abbreviations:
CAS = California Academy of Sciences, San Francisco, USA FMNH = Field Museum of Natural History, Chicago, USA ZFMK = Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute of Terrestrial Biodiversity, Bonn, Germany

Specimen collection and conservation
The described specimens of Sphaeromimus were all obtained from natural history collections and collected by hand during general inventory programs by American museums.

Dissection and illustrations
The left or the right 1 st , 2 nd and 9 th legs, the anterior and the posterior telopods of the male holotype, and the left or right 2 nd leg and the subanal plate of female paratypes (if present) were dissected for drawings.Pencil-drawings were made using a camera lucida mounted on a Zeiss Discovery V8 (Carl Zeiss AG).
For both species the antennae and a part of the endotergum of a mid-body segment were dissected for SEM-imaging.From the species found in Kalambatritra, where more specimens were available, the mandible and the gnathochilarium were dissected as well.All objects were dehydrated in an alcohol series (96%, 96%, 100%) and dried overnight.The parts were mounted on stubs and sputter-coated with gold.Objects were removed from the stubs and returned to alcohol after study.SEM-images were obtained with a Supra VR 300VP scanning electron microscope (Carl Zeiss AG) using the SmartSEM V05.00 software (Carl Zeiss AG).

Micro-CT imaging
For a non-invasive visualization of internal structures a micro-CT-scan was performed.The head of the female paratype of S. kalambatritra sp.nov.was dehydrated via an ascending ethanol series (96%, 100%) and critical point dried.A micro-CT-scan was conducted using the SKYSCAN 1272 micro-CT-scanner (Brucker microCT, Kontich, Belgium) and the accompanying Control Software v. 1.1.7(Brucker microCT) with the following settings: source voltage = 60 kV, source current = 166 μA , exposure = 915 ms, rotation of 180° in rotation steps of 0.2°, frame averaging = 6, random movement = 15, fl at fi eld correction ON, geometrical correction ON, fi lter = Al 0.25 mm.Reconstruction and thermal drift correction was performed in NRecon v. 1.7.0.4 (Brucker microCT) and the images were converted to 8bit grayscale images via Fiji ImageJ v. 1.49k (Schindelin et al. 2012).Volume rendering of the dataset for three-dimensional exploration and visualization was done in Drishti v. 2.6.3 (Limaye 2012).
The tentorium and nebententorium were segmented automatically and corrected manually in ITK-SNAP v. 3.4.0(Yushkevich et al. 2006).

DNA-extraction and sequencing
Muscle tissue was taken from the male holotypes stored in 80% ethanol and transferred to 95% ethanol.For DNA extraction the DNeasy Blood & Tissue Kit (Qiagen Sample & Assay Technologies) was used.A fragment of the cytochrome c oxidase subunit I (CO1) mitochondrial gene was amplifi ed with the primers HCO2198-JJ and LCO1490-JJ (Astrin & Stüben 2008) using the Multiplex PCR Kit (Qiagen Sample & Assay Technologies) and the Thermocycler Biometra TRIO (Biometra GmbH).The PCR protocol and other methods are the same as those used in earlier studies (Wesener 2015).The PCRproduct was enzymatically purifi ed with the Illustra ExoProStar PCR and Sequence Reaction Clean-MORITZ L. & WESENER T., Two new Sphaeromimus from Madagascar up Kit (GE Healthcare Life Sciences).Double-strand Sanger sequencing (Sanger et al. 1977) of the cleaned PCR product was performed by Macrogen, Netherlands.Sequences were concatenated using Seqman (DNASTAR Inc.).BLAST searches (Altschul et al. 1990) were used to check the sequences for contaminations.Sequences were translated into amino acids to rule out the accidental amplifi cation of pseudogenes.New sequences were uploaded to Genbank (see Table 1).

Sequence processing and DNA-analysis
Sequences were aligned by hand in Bioedit (Hall 1999).The fi nal dataset consisted of 33 sequences for 15 species; two of the sequences were newly sequenced and 31 obtained from GenBank (see Table 1), which had been part of two earlier studies (Wesener et al. 2010(Wesener et al. , 2014)).The fi nal dataset included 33 sequences with 674 positions.Distance analysis was performed in Mega 6 (Tamura et al. 2013) using the uncorrected p-distance model.Variation among sites was modeled with gamma distribution with shape parameter = 1.Included were codon positions 1 st +2 nd +3 rd .Genetic distances are displayed in matrix format (Table 2).The best fi tting substitution-model for maximum likelihood analysis was calculated with Modeltest (Tamura & Nei 1993) as implemented in Mega 6. Codon positions included were 1 st +2 nd +3 rd .The best fi tting model was the General Time Reversal (GTR)-Model (Tavaré 1986) with gamma distribution and Invariant sites (GTR+G+I) (lnL = -4010.400,Invariant = 0.5302, Gamma = 1.4196,R = 3.75520307816161; Freq A: 0.2640, T: 0.3345, C: 0.2287, G: 0.1726).Phylogenetic analysis was performed in Mega 6 based on the GTR+G+I model.A species tree was reconstructed using the maximum likelihood method with gamma-distribution of 5 categories.Initial trees were made automatically using NJ/BioNJ.The bootstrap consensus tree was calculated from 1000 replicates (Felsenstein 1985).The obtained tree was edited in Adobe Illustrator CS2.

Distribution map
The localities of specimens were mapped in the free software DIVA-GIS v. 7.5.0.0 (Hijmans et al. 2001) on spatial information made freely available by the GADM database v. 1.0 and the Madagascar Vegetation mapping project (Moat & Smith 2007).
COLORATION OF PRESERVED SPECIMEN (Fig. 1A).Tergites in anterior half dark brown, posterior half light brown, posterior margin with thin dark brown band.Paratergites light brown with dark brown to blackish tips.Paratergite impressions and groove of thoracic shield light brown.Antennae brown, and legs and pleurites light brown to grey.Head laterally around eyes and at posterior margin dark brown, frontally light brown.Collum dark brown.Eyes green.
HEAD.Eyes with >80 ocelli, median ocelli small and increasing in size towards lateral and posterior part of eyes (Fig. 1A), several larger ocelli on lateral margin clearly separated from eye.Organ of Tömösváry positioned in antennal-groove (Fig. 1B).Antennae short, protruding to coxa of third leg, not reaching margin of thoracic shield.Antennomere 1 as long as 2+3; antennomeres 2-5 of similar length; antennomere 6 as long as 4+5, but shorter than 1.Antennomeres 1-6 and antennal groove densely MORITZ L. & WESENER T., Two new Sphaeromimus from Madagascar pubescent (Fig. 2A).Antennomere 6 towards disc with single row of sensilla basiconica (Fig. 2B-C).Disc in female with 33/31, in male with 53/55 apical cones, as well as several setae shorter, or as long as, apical cones (Fig. 2B).Margin of labrum with setae.GNATHOCHILARIUM.Gnathochilarium typical for members of the order Sphaerotheriida, stipes and lamellamentum densely pubescent (Figs 1B, 3A, E).Lateral palpi rudimentary, not distinctly projecting over level of surrounding cuticle, consisting of four sensillae (Fig. 3C).Inner palpi well developed, with fi eld of sensory-cones and scale-like structures (Fig. 3D).Central pads (protuberance of endochilarium) with fi eld of sensory-cones and smaller scale-like structures (Fig. 3E).Endochilarium with deep triangular incision between central pads, central pads projecting lamella-mentum nearly to base of inner palpi.Lateral endochilarium with densely packed median-pointing setae.Hypopharynx with single row of teeth lateral on both sides, anterior distinctly separated row of 8 teeth (Fig. 3B).MANDIBLE.Mandible with typical shape of Sphaerotheriida (Fig. 3F), inner tooth 3-combed, with 6 long pectinate lamellae (Fig. 3G), condylus with a single, lower step at its apex (Fig. 3F).Condylus of mandible mounted against cuticular thickening on lateral margin of labrum, anteriorly of antennae (Fig. 1C).Tentorium lacking connection to head capsule via transverse bar.Posterior process triangular and plate-like, laying parallel to plate-like gnathal lobe sclerite.Epipharyngeal bar broad and laminar, running within epipahryngeal wall in direction of mandible condylus, broadening distally.Epipharyngeal bar with short lateral offshoot.Hypopharyngeal bar rod-like, curved within hypopharyngeal wall.Distal tip of hypopharyngeal bar reaches plate-like nebententorium.Nebententorium oriented at right angle to hypopharyngeal bar (Fig. 1E).
COLLUM.Collum glabrous except for few setae at margins.THORACIC SHIELD.Thoracic shield glabrous, with a chagrinated (leather-like) surface, few setae in grooves.Grooves deep (Fig. 1A).BODY RINGS.Tergites 3-12 with a chagrinated surface, small hair only present at posterior margin and in grooves, paratergite tips of mid-body tergites strongly projecting posteriorly (Fig. 1A).Tergites with single black locking carina.ANAL SHIELD.Anal shield large, with a steep edge, entirely glabrous, with lighter and darker patches (Fig. 1A).Underside with single black locking carina, located closer to tergite margins than to pleurite.ENDOTERGUM.Endotergum inner section with loose fi eld of short, cone-shaped spines and long setae (Fig. 4A).Externally 2-3 dense rows of long marginal bristles, slightly protruding above margin of tergite.Bristles covered with small, triangular spines, apically increasing in density (Fig. 4B).STIGMATIC PLATES.First stigma-carrying plate with a well-rounded projecting apex, apex covered with tiny spines and setae (Fig. 5A).Second plate (Fig. 5B) without apex and spines.
LEGS.Leg 1 with 2, 2 with 4, 3 with 10 ventral spines and no apical spine.Leg pairs 4-21 with 12-16 ventral spines and an apical spine.Small coxa process developed, covered with fi eld of spines (Fig. 5C).Femur 2.0, tarsus 3.3 times as long as wide.All podomeres with few setae (Fig. 5C).Toothed ridge (cleaning comb?) of femur with >40 teeth, reaching ca.0.8 times length of femur.Coxa in anterior aspect basally with a row of teeth, similar to "cleaning comb" on femur.Pronounced coxal process absent.MALE GONOPORE.Male gonopore typical for genus, plate glabrous, surrounded by relative large membranous area, few small spines basally of gonopore (Fig. 5B).5D).Shape usual for genus, with setae and tiny teeth at apical margin.Telopoditomere 4 with one large, triangular, apically weakly sclerotized spine and 2 smaller ones (Fig. 5E-F); basally on its anterior side with several tiny teeth.Telopoditomere 3 with triangular hump laterally, located close to border to telopoditomere 4 and juxtaposed to process of telopoditomere 2 (Fig. 5E).POSTERIOR TELOPOD.Podomere 3 slightly curved, 3.3 times as long as wide, slightly longer than immovable fi nger (Fig. 5G-H).Hollowed-out inner margin with two lobes and two spines, posterior aspect with ca 30 small crenulated teeth.Immovable fi nger straight, basally wide, apically tapering, only apical tip strongly curved towards podomere 3. Podomere 1 with few setae on lateral margin (Fig. 5H), podomere 2 only with few setae at anterior side, posterior side glabrous (Fig. 5G-H).Podomere 3 with only few marginal setae.FEMALE SEXUAL CHARACTERS.Vulva massive.Operculum well-rounded, protruding up to apical third of prefemur, with few marginal setae (Fig. 6A).Subanal plate large, with shallow invagination at apical margin (shape typical for genus).Washboard with ten stridulation ribs on each side, median in anterior half with black triangular fi eld (Fig. 6B).The female carried several hundred eggs with a diameter of 1.5-1.6 mm.

Distribution
Only known from the type-locality, the Réserve Spéciale de Kalambatritra, which is a mountainous rainforest (Fig. 11).In the same habitat, two undetermined giant pill-millipede species of the genus Zoosphaerium occur sympatrically.

Diagnosis
Large, massive, dark brown Sphaeromimus, >50 mm long.Differing from the only known species of Sphaeromimus with six stridulation ribs on the male harp (S. ivohibe Wesener, 2014), with which it also shares the two lobes on the movable fi nger of the posterior telopod, in the following characters: large difference in size and colour pattern, a densely pubescent male gonopore, legs 4-21 with 14 or 15 ventral spines (12 in S. ivohibe), and endotergum with two dense rows of long marginal bristles (single row in S. ivohibe).
COLORATION OF PRESERVED SPECIMEN.Tergites dark brown with black tips, posterior margin with a light brown band which is bordered anteriorly and posteriorly by thinner black bands.Paratergite impressions and groove of thoracic shield lighter brown.Legs, antennae, pleurites, head and collum brown, eyes green.
COLLUM.Collum glabrous except for few setae at margins.THORACIC SHIELD.Thoracic shield chagrinated (leather-like).Grooves deep, covered with setae.BODY RINGS.Paratergites 3-12 with posterior margin smooth, rest chagrinated, with hairs on posterior and anterior margin, paratergite tips of mid-body tergites strongly projecting posteriorly.ANAL SHIELD.Anal shield massive, well-rounded, lacking pubescent area.Underside with single black locking carina, located closer to tergite margins than to pleurite.ENDOTERGUM.Endotergum inner section with setae and triangular spines (Fig. 8A).Between margin and inner area with single row of large, elliptical cuticular patterns.Externally two row of marginal bristles.Bristles protruding weakly above tergite margin.Bristles with small triangular spines, apically increasing in density (Fig. 8B).STIGMATIC PLATES.First stigma-carrying plate with a well-rounded, projecting apex covered by hairs (Fig. 9A).Second plate without apex, but with fi eld of teeth opposite of coxa (Fig. 9B).

Female sexual characters
Unknown.

Distribution
Only known from the type-locality, the Parc National de Midongy (Fig. 11).Mountainous rainforest.

Barcoding results
S. kalambatritra sp.nov.shows the lowest genetic distance to the undetermined specimen from Vevembe (Fig. 11), with a 8.4% divergence.The distance of S. kalambatritra sp.nov. to S. midongy sp.nov.from the only rainforest located close-by is 9.4%.The highest distance of S. kalambatritra sp.nov.within Sphaeromimus is 19% to both S. inexpectatus and S. andohahela (Table 2).S. midongy sp.nov.also has the lowest pairwise distance to the undetermined Vevembe specimen, with 6.3%, and the highest to S. andohahela with 21.1% (Table 1).

Updated distribution of Sphaeromimus
Including the two newly described species, the genus Sphaeromimus is still restricted to southeastern Madagascar.With S. kalambatritra sp.nov., we record the western-most distribution of a rainforest species (Fig. 11).S. kalambatritra sp.nov.was, as all other species of Sphaeromimus, found to live in direct sympatry with at least one species of Zoosphaerium.

Micro-CT in taxonomy and phylogeny
Already Akkari et al. (2015) showed the great potential of micro-CT in millipede taxonomy.It can be used to visualize and infer internal morphological characters, which can be exploited not only for the species description itself, but also for subsequent morphological phylogenetic reconstruction as demonstrated by Blanke & Wesener (2014).Here, we show that micro-CT is not only a useful tool to examine hard sclerotized structures like the head capsule, the gnathal lobe sclerite and the tentorium (Fig. 1C), but also to visualize soft parts like the collagenous tendon and single muscles (Fig. 1D).The advantage of micro-CT compared to conventional invasive methods is that it does not damage the specimen.This is especially important, because many new species are found in museum collections and are only represented by a few or a single specimen, like the species described in this study.These specimens cannot be examined without permission and should not be damaged.This makes it nearly impossible to study internal features by dissection or histological sectioning.Furthermore, it makes it possible to share morphological information via public databases, which otherwise opens up a variety of new possibilities, as outlined by Akkari et al. (2015).Therefore, we suggest considering the use of micro-CT in taxonomic studies if it seems advantageous to include internal characters.For the fi rst time we add internal characters to the taxonomic description of a member of the order Sphaerotheriida.We describe the structure of the tentorium and the articulation of the mandible condylus.At this point, it is not possible to state which of these characters show inter-or intraspecifi c variations, because there is no data available yet for other species, except the descriptions of the tentorium of Arthrosphaera dentigera (Verhoeff 1930) by Verhoeff (1932) and Sphaeropoeus modiglianii Silvestri, 1895, a Zephroniidae, by Silvestri (1903).Future studies will elaborate whether any useful taxonomic characters can be collected from these inner morphological features.The structure of the tentorium of S. kalambatritra sp.nov.corresponds to the state described for A. dentigera.The hypopharnygeal bar of the tentorium of Sphaeromimus is elongated, while the nebententorium is more stout compared to the state depicted by Silvestri (1903) for the Zephroniidae.Furthermore, the posterior process of Sphaeromimus seems larger than in Sphaeropoeus, although this could be due to the perspective Silvestri chose to depict the tentorium.The shape of the epipharyngeal bar shows a high similarity in both.

Genetic analysis
The high genetic distances between the species of Sphaeromimus suggest an early speciation event, as previously suggested by Wesener et al. (2010Wesener et al. ( , 2014)).The COI gene probably lost its resolution due to the accumulation of reverse substitutions over time.Klopfstein et al. (2010) and Townsend & Leuenberger (2011) showed that the 'informativeness' of the COI gene declines in older splits.
As shown in several studies on giant pill-millipedes (e.g., Wesener et al. 2010 for Sphaeromimus; Wongthamwanich et al. 2012 andGolovatch et al. 2012 for Zephroniidae), the COI gene remains nevertheless a powerful tool for the taxonomy and identifi cation of giant pill-millipede species.It makes it possible to assign specimens of different stages, gender or with high morphological intraspecifi c variation to a species.Otherwise, it is not necessarily possible to assign such specimens to a species based solely on morphological evidence (see Wesener et al. 2014).The COI barcode shows a close and well-supported relationship between S. midongy sp.nov.and the undetermined (female) specimen from Vevembe (Fig. 11).In previous studies, the specimen from Vevembe was separated from S. ivohibe + S. musicus by a long branch and with weak support (see Wesener et al. 2014).With a genetic distance of 6.3% between S. midongy sp.nov.and the specimen from Vevembe, they cannot be assigned to the same species based on COI data alone until morphological characters of the male of the Vevembe population become available.Thus, S. inexpectatus and S. saintelucei show a genetic distance of only 4.1% in our analysis and 4.0% in the study conducted by Wesener et al. (2014), and are regarded as separate species based on morphological characters.Nevertheless, the genetic distance of the COI sequence fragment in Sphaeromimus is usually 8-20% between two species (Wesener et al. 2014).Therefore, it cannot be ruled out that the specimen from Vevembe belongs to S. midongy sp.nov.To draw suffi ciently supported conclusions, further studies and sampling of these areas are needed.A comparison of the genetic distance and the geographical distance reveal an interesting biogeographic pattern.S. ivohibe and the specimen from Vevembe, which are separated by ca 43 km (Fig. 11), show a genetic distance of 15.1% while S. midongy sp.nov.and the specimen from Vevembe are separated by 125 km (Fig. 11), and have a genetic distance of 6.3%.The genetic distance between S. midongy sp.nov.and S. kalambatritra sp.nov.MORITZ L. & WESENER T., Two new Sphaeromimus from Madagascar is 9.4%, although the geographical distance is only 65 km (Fig. 11).This demonstrates that species endemic to geographically close habitats are not necessarily closely related (see also Wesener et al. 2014).

Number of stridulation ribs of the male harp
Comparing the number of stridulation ribs on the male harp to the species-tree, there seems to be one clade with an increased number of stridulation ribs (Fig. 10 European Journal of Taxonomy 381: 1-25 (2017) to be noted that there is no information on the number in the specimen from Vevembe, since we know a single female only.We can assume that the ancestral state is a low number of stridulation ribs on the male harp, as seen in other representatives of the family Arthrosphaeridae.In Arthrosphaera the number of stridulation ribs can vary between none and 2 ribs, Microsphaerotherium has one rib and Zoosphaerium has one or two (Wesener & VandenSpiegel 2009).The advantage of a high number of stridulation ribs remains unclear.Thus, the habitats of species with high or low numbers of stridulation ribs overlap.Species of Sphaeromimus and Zoosphaerium usually occur in sympatry (Wesener et al. 2014).The number of ribs might have an impact on the sound production, but only for South African species information about the produced sounds is available (Wesener et al. 2011).
Fig. 10.Maximum likelihood tree based on the COI sequence after 1000 bootstrap replicates under the GTR+G+I model.Branch length indicates genetic distance.Numbers on branches indicate bootstrap support.Schematic drawing shows right anterior telopod of Sphaeromimus midongy sp.nov.Numbers in circles indicate number of stridulation ribs on the male harp.
The single specimen of the species collected in Midongy (S. midongy sp.nov.) by V. Soramalaia in February 2008, was provided by the FMNH.The four specimens from Kalambatritra (S. kalambatritra sp.nov.; Fig. 2A), collected by B.L. Fisher and colleagues in February 2009, are from the collection of the CAS.