Dichromatobolus, a new genus of spirobolidan millipedes from Madagascar (Spirobolida, Pachybolidae)

A new genus, Dichromatobolus gen. nov., belonging to the genus-rich mainly southern hemisphere family Pachybolidae of the order Spirobolida, is described based on D. elephantulus gen. et sp. nov., illustrated with color pictures, line drawings, and scanning electron micrographs. The species is recorded from the spiny bush of southwestern Madagascar. Dichromatobolus elephantulus gen. et sp. nov. shows an unusual color pattern, sexual dichromatism with males being red with black legs and females being grey. Males seem to be more surface active, as mainly males were collected with pitfall traps. Females mainly come from the pet trade. The body of this species is short and very wide, being only 8 times longer than wide in the males. Live observations show the species is a very slow mover, digging in loose soil almost as fast as walking on the surface. The posterior gonopods of Dichromatobolus gen. nov. are unusually simple and well-rounded, displaying some similarities to the genera Corallobolus Wesener, 2009 and Granitobolus Wesener, 2009, from which the new genus diff ers in numerous other characters, e.g., size, anterior gonopods and habitus. Despite several attempts with fresh tissue samples and diff erent primers, molecular barcoding did not work for Dichromatobolus gen. nov. Any relationships to the other 15 genera of Pachybolidae indigenous to Madagascar remain unknown.


Introduction
Madagascar, the large island off the eastern coast of Africa, is famous for being a biodiversity hotspot (Myers et al. 2000), as well as a center of endemism for numerous animal and plant groups (Goodman & Benstead 2005;Pearson & Raxworthy 2009). Because of differences in rainfall and altitude (Battistini 1972;Jury et al. 1995;Dewar & Richard 2007), Madagascar hosts several different ecosystems, from spiny dry forests to montane rainforests (Moat & Smith 2007), probably the main drive behind an extraordinary species richness in several groups of organisms. Millipedes (class Diplopoda de Blainville, 1844) are no exception, with most known species being endemic to Madagascar (Enghoff 2003), and numerous known microendemic species (Wesener & Sierwald 2005;Wesener 2009;Wesener et al. 2014;Moritz & Wesener 2017). Only six of the 16 (Blanke & Wesener 2014) orders of the Diplopoda are known with indigenous described species from Madagascar, but two more, Polyzoniida Gervais, 1844 (Wesener 2014a) and Siphonophorida Newport, 1844 (Wesener 2014b), are present with indigenous, but still undescribed species. Especially rich in genera and species on Madagascar is the family Pachybolidae Cook, 1897 sensu Hoffman 1980(order Spirobolida Cook, 1895  ). Altogether 15 of the 16 indigenous genera of the Spirobolida on Madagascar belong to the Pachybolidae, with only Hylekobolus Wesener, 2009 belonging to the family Spirobolellidae Brölemann, 1913. With 15 currently described genera, Madagascar is also one of the main centers of diversity of the Pachybolidae globally, a family showing a Gondwanan distribution (Enghoff et al. 2015). The relationships of the Malagasy genera of the Pachybolidae are still unclear. One genus, Madabolus Wesener & Enghoff, 2008, occurring in western Madagascar is clearly, based on morphological (Wesener et al. 2008) and genetic characters (Pitz & Sierwald 2010), a member of the tribe Pachybolini Wesener & Enghoff, 2008, which is further distributed in western and eastern Africa. Another genus Spiromimus de Saussure & Zehntner, 1901, diverse in dry forests and rainforests in the northern half of Madagascar, shows some morphological similarities with the genus Xenobolus Carl, 1919 from southern India ). The relationships of the other 13 genera of the Pachybolidae endemic to Madagascar are still unclear and might be only resolved with a comprehensive molecular study including other genera from South America, South Africa, India, Southeast Asia and Australia. At the species level, many species of Pachybolidae are restricted to very small areas of distribution (e.g., Wesener 2011;Wesener et al. 2011), and 25 of the 58 known endemic species are listed in one of the top three IUCN Red List categories (e.g., Rudolf & Wesener 2017a, 2017b, 2017c, 2017d.
Here we describe a new genus from Madagascar, based on a new species from the southwestern spiny bush featuring unusual morphological characters.

CAS
= California Academy of Sciences, San Francisco, USA CASENT = Entomology collection, California Academia of Sciences, San Francisco, USA FMNH = Field Museum, Chicago, USA ZFMK = Zoological Research Museum A. Koenig, Leibniz Institute for Animal Biodiversity, Bonn, Germany

Illustrations
Dissecting and camera lucida drawings were done under an Olympus SZX12 stereo microscope. For scanning electron microscopy (SEM), the samples were dehydrated via an ethanol chain, mounted on stubs and dried overnight. The stubs were 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. The locality of specimens was mapped in the free software DIVA-GIS 7.5.0.0 (Hijmans et al. 2001) on spatial information made freely available by the GADM database 1.0 and the Madagascar Vegetation mapping project (Moat & Smith 2007).

Collection and deposition
No specimens were collected by the author, they come mainly from the backlog of the FMNH and CAS. An additional large amount of specimens (from unknown localities) came from the pet trade, both from an import in 2007 by the German firm (now no longer in existence) Odenwald Exoten, and from an additional import in 2019 (only males) by the French trader ebugz.eu. Specimens were received for identification and are stored in the public museum collections of the CAS, FMNH and ZFMK.

DNA extraction attempts
Despite four DNA extractions, and the utilization of PCR protocols which have yielded good sequence data for the Spirobolida ) and other millipedes (Wesener 2015a(Wesener , 2015b in two laboratories, the Field Museum, Chicago, USA and the ZFMK, no good COI barcoding sequences could be obtained. Utilized primer pairs for PCR and sequencing were the HCO / LCO primers (Folmer et al. 1994) and modified degenerated primers, HCO-JJ / LCO-JJ (Astrin & Stüben 2008). Curiously, something not observed in any of the other Diplopoda COI sequences we analyzed so far, the PCR result showed triple bands, and the sequencing result indicated contaminations.

Diagnosis
Massive, short, differs from all other known genera of Pachybolidae in mature males being only eight times longer than wide. Coloration uniquely differing between sexes, females grey, males red (

Etymology
After the differences in the color pattern between the sexes, and ʻ-bolusʼ, a common suffix for genus names of the Spirobolida. Masculine.

Taxonomic position
The posterior gonopods being connected by a sclerotized sternite as well as the absence of apodemes on the anterior gonopods ( Fig. 4E-F) place Dichromatobolus gen. nov. in the suborder Trigoniulidea Attems, 1909, family Pachybolidae. The closest relative of Dichromatobolus gen. nov. is unknown.

Remarks
There is a second potential species, > 100 mm in length, with specimens from Manongarivo, Ananambao (FMNH-INS 56254), all unfortunately female. Their wide body with short legs and grey color (all other larger Spirobolida genera on Madagascar are black / red,   Coloration. Sexually dimorphic (Fig. 1A). Male: head and body reddish (Fig. 1A-E), telson bright red (Fig. 1F), ozopores marked by black spot. Legs and antennae black (Fig. 1B-F). Female: Head and body including telson grey (Fig. 1A), ozopores marked by black spot. Posterior margin of body rings darker. Legs and antennae black.
Body rings. Ozopores marked by black color, starting at ring 6, located a half diameter before suture between mesozona and metazona. Rings with smooth, but irregular coriaceous surface, ventrally on metazona with transverse ridges. Limbus simple, with cuticular scales and wavy margin (Fig. 3A).
Legs. Leg 1 with a large cylindrical coxa, twice as long as other podomeres (Fig. 4C). Tarsus with three pairs of ventral spines, without an apical spine beyond claw. Leg 2 with a strongly elongated coxa, fused to sternite. Tarsus with four pairs of ventral spines and a short apical spine (Fig. 4B). Midbody legs with a rectangular coxa, as long as other podomeres. Each podomere ventrally with a single or a pair of apical setae, tarsus in males with a ventral pad, 6 pairs of ventral and 5 pairs of lateral spines (Fig. 4D). Length of midbody legs circa 0.7 times body diameter in males (Fig. 1E).

Female sexual characters
Body color grey (Fig. 1A). Female vulva simple, resembling a bivalve shell.

Intraspecific variation
The three known populations from southwestern Madagascar, as well as the specimens from the pet trade, coming from at least two different imports 2007 and 2019 look very similar to one another, showing only a slight variation in body ring counts, varying between 47 and 48.

Remarks
Some element associated with the species, or the surroundings they live in, reacts to ethanol. White crystallizations appear around the specimens and appendages when conserved in ethanol for a longer time.

Life observations
The fact that this species was part of the pet trade allowed a few life observations. Dichromatobolus elephantulus gen. et sp. nov. did not last long in captivity. Specimens of both the 2007 and the 2019 import died after 2-3 weeks. Males and females of the 2007 import were imported as two different species because of the difference in the color pattern. Such a strong color differences observed between the sexes as in D. elephantulus gen. et sp. nov. have to our knowledge not been reported in any other Juliformia species. Sexual color dimorphism is known for some species of Megaphyllum Verhoeff, 1894 of the related order Julida Brandt, 1833 (Schubart 1934). Also, in the Spirobolida, males are often of a shinier appearance compared to females, as in the African species Epibolus pulchripes (Gerstäcker, 1873), as was illustrated in a recent developmental article (Dhaenens & VandenSpiegel 2006). In 2019, only males could be observed. Specimens of both sexes were very sluggish, not moving after a disturbance like touching. When moving on the surface they were slow. Digging into loose substrate was as fast as walking on the surface. Specimens of both sexes tried to walk around, not climb up obstacles. Females of the 2007 import were strongly infected with mites, which covered the anterior part of the body and all of the legs. Mites were also present in some of the males (Fig. 3B). The illustrated mite was determined by one of the reviewers as a hypopi, phoretic deutonymphs of acaridian mites.

Outlook
The discovery of Dichromatobolus gen. nov. shows how little we still know about the diversity of the Pachybolidae on Madagascar, despite the fact that the number of known endemic genera of the island grew from two before 2008 to 16 now. The spiny forest ecosystem, with the great seasonality of rainfall, might make any discovery of surface active millipedes quite difficult, as the roads are difficult to traverse during the rainy season when the millipedes are most likely active on the surface and sexually mature.